Circular RNA Synthesis

Abstract

This disclosure provides a genetic construct (DNA or RNA) that can be used to generate circular RNA. In some aspects, the genetic construct containing group I introns (e.g., precursor cirRNA or DNA transcribed into precursor cirRNA) can be combined with GTP or allowed to self-circularize to generate circular RNA.

Description

Cross-reference to related applications

[0001] This PCT application claims priority to U.S. Provisional Application No. 63 / 601,185, filed November 20, 2023; the entire disclosure of which is incorporated herein by reference for all purposes.

[0002] References to sequence lists submitted electronically via EFS-WEB The contents of the sequence list are submitted electronically (name:; size: bytes; and creation date:) and are filed together with the application, which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to methods and compositions for generating circular RNA (circRNA). Background Technology

[0004] Circular RNA (circRNA) is a covalently closed single-stranded RNA molecule that lacks free 5' and 3' ends, and therefore lacks a 5' cap and a 3' polyA tail.

[0005] In nature, circRNAs have a wide range of sources and tissue specificity, and play a variety of roles in the development and homeostasis of organisms, as well as in aging. They are also associated with the occurrence and development of various human diseases, such as cardiovascular disease, diabetes and cancer.

[0006] circRNAs are more stable than linear RNAs, do not require a 5' cap or 3' poly(A) to maintain their stability, and are not easily degraded by RNA exonucleases (Petkovic, S., and Müller, S. (2013). RNA Self-Processing: Formation of Cyclic Species and Concatemers from a Small Engineered RNA. FEBSLett. 587, 2435–2440.), making circRNAs excellent candidates as therapeutic agents.

[0007] However, the use of circRNA as a therapeutic agent is hampered by technical limitations in its synthesis.

[0008] Linear RNA can be produced through chemical synthesis or enzymatic strategies, and circRNA can be produced by ligating the ends of a linear RNA precursor to create a covalently closed circular structure. The use of chemical synthesis is limited by high purification costs and low yields, and can only produce RNA shorter than 50 to 70 nucleotides. Enzymatic strategies are typically achieved through in vitro transcription (IVT) reactions, which involve a DNA template, a reaction buffer, and phage RNA polymerase. IVT reactions allow for the synthesis of longer RNAs at a lower cost. However, the run-off nature of phage polymerase can result in incomplete RNA.

[0009] Ribozyme methods include the group I intron self-splicing system, also known as the rearranged intron and exon (PIE) method. Due to the complexity of RNA secondary structure, different RNA sequences lead to differences in splicing efficiency, and thus to differences in the final circRNA yield, which limits the application of the PIE method. Group II introns can also be used for circRNA synthesis, which involves a reverse splicing reaction. This method forms a 2',5'-phosphodiester bond at the linker site instead of the native 3',5'-phosphodiester bond, and the mechanism remains unclear. Hairpin ribozymes (HPRs) can generate circRNA from circular single-stranded DNA templates via rolling circle reactions and self-splicing reactions. However, the HPR method can only efficiently produce small circRNAs and has unstable products and exogenous HPR sequences.

[0010] Furthermore, the high immunogenicity of synthetic circRNAs (Chen, YG et al., (2017). Sensing self and foreign circular RNAs by intron identity. Mol. Cel. 67, 228–238.e5; Chen, YG et al. (2019). N6-methyladenosine modification controls circular RNAimmunity. Mol. Cel. 76, 96–109.e) further hinders the applicability of circRNAs as therapeutic molecules.

[0011] Given the strong therapeutic potential of circRNA, there is an urgent need for methods and compositions for the efficient production of circRNA and for the production of circRNA with low immunogenicity. Summary of the Invention

[0012] The present disclosure addresses the aforementioned and / or other problems. In some aspects, the present disclosure provides a genetic construct comprising the following formula: 5'-HAEB-H'-3', Wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence (IGS), and H' contains a 3' homologous arm, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site, and wherein the genetic construct is capable of forming a circular RNA, the circular RNA not containing any exon sequences ("excision scars") derived from the genome from which the 5' intron sequence and the 3' intron sequence are derived.

[0013] In some respects, the 5' intron sequence and / or the 3' intron sequence are derived from group I introns. In some respects, the 3' intron sequence and the 5' intron sequence together are smaller than the complete intron.

[0014] In some respects, the 5' intron sequence and / or the 3' intron sequence are derived from introns derived from: thymidine synthase (td) from T4 phage; or from the cyanobacterium *Azolla*. (Anabaena azollae) Anabaena (Anabaena) PCC7120, Alternaria flocculation (Aphanizomenon flos-aquae) strain NIVA-CYA 142 or Synechococcus (Synechococcus) Pre-tRNA LEU Nitrogen-fixing bacteria, Vibrio azotocinus (Azoarcus) Pre-tRNA ILE From Tetrahymena thermophila (Tetrahymena thermophila) 26S ribosomal RNA; from Papilionophora. (Trimorphomyces papilionaceus) of COX2 Genes; or any combination thereof.

[0015] In some aspects, the 5' intron sequence and / or the 3' intron sequence are derived from introns of thymidine synthase (td) from T4 phage. In some aspects, the 5' splice site comprises a site between two nucleotides containing U and N, where N is any nucleotide. In some aspects, N is A, T, G, or C. In some aspects, the 3' splice site comprises a site between two nucleotides containing G and N, where N is any nucleotide. In some aspects, N is C. In some aspects, the catalytic core comprises ACG, CU, or both.

[0016] In some aspects, the excision scar comprises SEQ ID NO: 80 or SEQ ID NO: 82. In some aspects, the excision scar comprises both SEQ ID NO: 80 and SEQ ID NO: 82. In some aspects, the excision scar comprises either SEQ ID NO: 81 or SEQ ID NO: 83. In some aspects, the excision scar comprises both SEQ ID NO: 81 and SEQ ID NO: 83.

[0017] In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 3' intron sequences (SEQ ID NO: 11–22) selected from Table 4. In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 5' intron sequences (SEQ ID NO: 23–30) selected from Table 4.

[0018] In some respects, the 5' intron sequence and / or the 3' intron sequence are derived from the cyanobacterium *Azolla*. tRNA LEU The introns. In some aspects, the 5' splice site comprises a site between two nucleotides containing U and N, wherein N is any nucleotide. In some aspects, N is A. In some aspects, the 3' splice site comprises a site between two nucleotides containing G and N, wherein N is any nucleotide. In some aspects, N is C. In some aspects, the catalytic core comprises GAG, CC, or both. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 3' intron sequences in Table 4, such as SEQ ID NO: 14 or 20. In some respects, the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 5' intron sequences in Table 4, such as SEQ ID NO: 25 or 29.

[0019] In some respects, the 5' intron sequence and the 3' intron sequence are derived from the genus *Vibrio zonatogenes*. tRNA LEU The introns. In some aspects, the 5' splice site comprises a site between two nucleotides containing U and N, wherein N is any nucleotide. In some aspects, N is A. In some aspects, the 3' splice site comprises a site between two nucleotides containing G and N, wherein N is any nucleotide. In some aspects, N is G. In some aspects, the catalytic core comprises GAG, CC, or both. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 3' intron sequences in Table 4, such as SEQ ID NO: 13 or 19. In some respects, the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 5' intron sequence in Table 4 (e.g., nucleotide sequence AAA).

[0020] In some aspects, the 5' intron sequence and the 3' intron sequence are derived from introns of 26S ribosomal RNA from Tetrahymena thermophila. In some aspects, the 5' splice site comprises a site between two nucleotides containing U and N, wherein N is any nucleotide. In some aspects, N is A. In some aspects, the 3' splice site comprises a site between two nucleotides containing G and N, wherein N is any nucleotide. In some aspects, N is U. In some aspects, the catalytic core comprises a catalytic core selected from Table 5. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 3' intron sequences in Table 4, such as SEQ ID NO: 12 or 18. In some respects, the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 5' intron sequences in Table 4, such as SEQ ID NO: 24 or 28.

[0021] In some aspects, this disclosure provides a genetic construct comprising the formula: 5'-HAEB-H'-3', Wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence (IGS), and H' contains a 3' homologous arm, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site, wherein the genetic construct is capable of forming a circular RNA, wherein the circular RNA does not contain any exon sequence ("excision scar") derived from the genome from which the 5' intron sequence and the 3' intron sequence are derived, and wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 113 or 123.

[0022] In some aspects, this disclosure provides a genetic construct comprising the formula: 5'-HAEB-H'-3', Wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence (IGS), and H' contains a 3' homologous arm, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site, wherein the genetic construct is capable of forming a circular RNA, wherein the circular RNA does not contain any exon sequence ("excision scar") derived from the genome from which the 5' intron sequence and the 3' intron sequence are derived, and wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 114 or 124.

[0023] In some aspects, this disclosure provides a genetic construct comprising the formula: 5'-HAEB-H'-3', Wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence (IGS), and H' contains a 3' homologous arm, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site, wherein the genetic construct is capable of forming a circular RNA, wherein the circular RNA does not contain any exon sequence ("excision scar") derived from the genome from which the 5' intron sequence and the 3' intron sequence are derived, and wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 115 or 125.

[0024] In some aspects, this disclosure provides a genetic construct comprising the formula: 5'-HAEB-H'-3', Wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence (IGS), and H' contains a 3' homologous arm, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site, wherein the RNA construct is capable of forming a circular RNA, wherein the circular RNA does not contain any exon sequence ("excision scar") derived from the genome from which the 5' intron sequence and the 3' intron sequence are derived, and wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 116 or 126.

[0025] In some aspects, this disclosure provides a genetic construct comprising the formula: 5'-HAEB-H'-3', Wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence (IGS), and H' contains a 3' homologous arm, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site, wherein the genetic construct is capable of forming a circular RNA, wherein the circular RNA does not contain any exon sequence ("excision scar") derived from the genome from which the 5' intron sequence and the 3' intron sequence are derived, and wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 117 or 127.

[0026] In some aspects, this disclosure provides a genetic construct comprising the formula: 5'-HAEB-H'-3', Wherein, H contains a 3' homologous arm, A contains a 3' homologous arm and a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence, and H' contains a 5' homologous arm. The 3' intron sequence contains a catalytic core. The 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site. The 5' intron sequence and / or the 3' intron sequence are derived from the precursor tRNA of the cyanobacteria *Azotobacter spp.*, *Vibrio spp.*, or *Synechococcus*. LEU It may be derived from the 26S ribosomal RNA of Tetrahymena thermophila and is capable of forming circular RNA.

[0027] In some respects, the 5' intron sequence and / or the 3' intron sequence are derived from the tRNA of the cyanobacterium *Azolla*. LEUIn some aspects, the 5' splice site comprises a site located between two nucleotides comprising U and any nucleotide (N), optionally A. In some aspects, the 3' splice site comprises a site located between two nucleotides comprising G and any nucleotide (N), optionally C. In some aspects, the catalytic core comprises GAG, CC, or both. In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 25 or 29. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 14 or 20.

[0028] In some respects, the 5' intron sequence and / or the 3' intron sequence are derived from tRNAs of the cyanobacterium Vibrio genus. LEU In some aspects, the 5' splice site comprises a site between two nucleotides comprising U and any nucleotide, optionally A. In some aspects, the 3' splice site comprises a site between two nucleotides comprising G and any nucleotide, optionally C. In some aspects, the catalytic core comprises GAG, CC, or both. In some aspects, the 5' intron sequence comprises a nucleotide sequence of AAA. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 13 or 19.

[0029] In some respects, the 5' intron sequence and the 3' intron sequence are derived from Synechococcus tRNA. LEUIn some aspects, the 5' splice site comprises a site located between two nucleotides containing U and any nucleotide (N), optionally A. In some aspects, the 3' splice site comprises a site located between two nucleotides containing G and any nucleotide (N), optionally C. In some aspects, the catalytic core comprises GAG, CC, or both. In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 5' intron sequence derived from Synechococcus introns, as shown in SEQ ID NO: 140 or 143. In some respects, the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 5' intron sequence derived from Synechococcus introns, as shown in SEQ ID NO: 140 or 143.

[0030] In some aspects, the 5' intron sequence and the 3' intron sequence are derived from the 26S ribosomal RNA of Tetrahymena thermophila. In some aspects, the 5' splice site comprises a site located between two nucleotides containing U and any nucleotide (N), optionally A. In some aspects, the 3' splice site comprises a site located between two nucleotides containing G and any nucleotide (N), optionally C. In some aspects, the catalytic core comprises GAG, CC, or both. In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 24 or 28. In some respects, the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 12 or 18.

[0031] In some respects, the circular RNA does not contain excision scars. In some respects, the circular RNA has reduced immunogenicity. In some respects, the circular RNA has reduced immunogenicity compared to circular RNA containing the excision scars.

[0032] In some aspects, this disclosure provides a genetic construct comprising the formula: 5'-HAEB-H'-3', Wherein, H contains a 3' homologous arm, A contains a 3' homologous arm and a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence, and H' contains a 5' homologous arm. The 3' intron sequence contains a catalytic core. The 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site. The 5' intron sequence and / or the 3' intron sequence are derived from the precursor tRNA of the cyanobacteria *Azotobacter spp.*, *Vibrio spp.*, or *Synechococcus*. LEU Or derived from 26S ribosomal RNA of Tetrahymena thermophila and capable of forming circular RNA, and said genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 118 or 128.

[0033] In some aspects, this disclosure provides a genetic construct comprising the following formula: 5'-AE-3', Wherein A contains a 3' intron sequence, E contains an exon sequence, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splicing site, and wherein the genetic construct is capable of forming circular RNA.

[0034] In some aspects, this disclosure provides a genetic construct comprising the following formula: 5'-AE-3', Wherein A contains a 3' intron sequence, and E contains an exon sequence, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 119 or 129, and wherein the genetic construct is capable of forming a circular RNA.

[0035] In some aspects, this disclosure provides a genetic construct comprising the following formula: 5'-AE-3', Wherein A contains a 3' intron sequence containing a catalytic core (CC), and E contains an exon sequence containing a first portion (E'); a first homologous arm (HI'); a second portion (E''); a second homologous arm (HI''); and a third exon sequence containing a catalytic nucleotide (CN) (E'''); wherein the 3' end of A and the 5' end of E form a 3' splice site, wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 120 or 130, and wherein the genetic construct is capable of forming a circular RNA.

[0036] In some aspects, this disclosure provides a genetic construct comprising the following formula: 5'-AE-3', Wherein A contains a 3' intron sequence containing a catalytic core (CC), and E contains an exon sequence containing a first portion (E'); a first homologous arm (HI'); a second portion (E''); a second homologous arm (HI''); and a third exon sequence containing a catalytic nucleotide (CN) (E'''); wherein the 3' end of A and the 5' end of E form a 3' splice site, wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 121 or 131, and wherein the genetic construct is capable of forming a circular RNA.

[0037] In some aspects, this disclosure provides a genetic construct comprising the following formula: 5'-AE-3', Wherein H contains a 3' homologous arm, A contains a 3' intron sequence containing a catalytic core (CC), and E contains an exon sequence containing a first portion (E'); a first homologous arm (HI'); a second portion (E''); a second homologous arm (HI''); and a third exon sequence containing a catalytic nucleotide (CN) (E'''); wherein the 3' end of A and the 5' end of E form a 3' splice site, wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 122 or 132, and wherein the genetic construct is capable of forming a circular RNA.

[0038] In some aspects, this disclosure provides a genetic construct comprising the following formula: 5'-AE-3', Wherein A comprises a 3' intron sequence containing a catalytic core (CC), and E comprises an exon sequence containing a first portion (E'); a first homologous arm (HI'); a second portion (E''); a second homologous arm (HI''); and a third exon sequence containing a catalytic nucleotide (CN) (E'''); wherein the genetic construct comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 181, and wherein the genetic construct is capable of forming a circular RNA.

[0039] In some aspects, this disclosure provides a genetic construct comprising the formula: 5'-BAE-3', Wherein A contains a 3' intron sequence, 3S contains a 3' splice site, E contains an exon sequence, B contains a 5' intron sequence, and 5S contains a 5' splice site, wherein the 3' intron sequence contains a catalytic core, wherein BA contains an intact intron, wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 182 or 183, and wherein the genetic construct is capable of forming a circular RNA.

[0040] In some respects, the genetic construct is RNA. In other respects, the genetic construct is DNA.

[0041] In some aspects, the genetic construct also includes a promoter. In some aspects, the promoter is a T7 promoter, a T3 promoter, or a Sp6 promoter.

[0042] In some respects, the genetic construct also includes IRES.

[0043] In some respects, the genetic construct also includes a polyA-binding protein binding site at its 5' end.

[0044] In some aspects, the 5' homologous arm comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same nucleotide sequence as SEQ ID NO: 72 (CCGTCGATTGTCCACTGGT). In some aspects, the 3' homologous arm comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same nucleotide sequence as SEQ ID NO: 73 (ACCAGTGGACAATCGACGG).

[0045] In some aspects, the exon sequence is connected to one or more untranslated regions at the 3' and / or 5' of the exon sequence. In some aspects, the one or more untranslated regions contain an IRES. In some aspects, the IRES contains a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 87-110 or 145-168. In some aspects, the IRES is connected to a polyA. In some aspects, the one or more untranslated regions contain a polyA. In some aspects, the exon sequence is connected to a polyA. In some aspects, the polyA contains a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 85 or 86. In some aspects, the one or more untranslated regions also contain a spacer sequence. In some aspects, the spacer region contains a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 74-75 or 78-79.

[0046] In some aspects, the exon sequences encode proteins. In some aspects, the circular RNA exhibits increased expression of the protein, prolonged expression of the protein in vitro and / or in vivo, decreased activation of immune signaling pathways, increased immune activation, or any combination thereof. In some aspects, the protein comprises a therapeutic protein. In some aspects, the protein comprises a cytokine, receptor, ligand, immunomodulator, growth factor, antigen, RNA-binding domain, or any combination thereof. In some aspects, the protein comprises an antibody, a fusion protein, or any combination thereof. In some aspects, the proteins include CD3, CD4, CDS, CD19, CD20, CD22, CD30, CD34, HER2, HER3, HER4, LFA-I, MoI, p150, 95, VLA-4, ICAM-I, VCAM, αv / β3 integrin, human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), NGF-β, platelet-derived growth factor (PDGF), fibroblast growth factor, epidermal growth factor (EGF), transforming growth factor (TGF), insulin-like growth factor-I and-II (IGF-I and IGF-II), des(l-3)-IGF-I (Brain IGF-1), Factor VIII, Tissue Factor, von Willebrand Factor, Protein C, α-1-antitrypsin, plasminogen activator such as urokinase and tissue plasminogen activator (“t-PA”), bufotoxin, thrombin, thrombopoietin, M-CSF, GM-CSF, G-CSF, flk2 / flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptor, thrombopoietin receptor (“TPO-R”, “c-mpl”), glucagon receptor, interleukin receptor, interferon receptor, T cell receptor, stem cell factor receptor, OX40L, bone-derived neurotrophic factor (BDNF), neurotrophic factor-3,-4,-5 or-6 (NT-3, NT-4, NT-5, or NT-6), relaxin A chain, relaxin B chain, pro-relaxin, interleukins and interleukin receptors such as IL-8 receptor including IL-1 to IL-33 and IL-1 to IL-33 receptors, viral antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-α and-β, enkephalin, RANTES (regulatory activation of normal T-cell expression of secretory factors), mouse gonadotropin-related peptide, DNase, inhibin, activin, PUF domain, Cas protein, or any combination thereof.

[0047] In some aspects, this disclosure provides a composition comprising the genetic construct. In some aspects, the composition comprises the genetic construct disclosed herein and GTP. In some aspects, the amount of GTP in the composition is sufficient to cleave the 5' intron sequence and the 3' intron sequence from the genetic construct.

[0048] In some aspects, this disclosure provides a method for preparing circular RNA, which includes contacting GTP with the genetic construct disclosed herein.

[0049] In some respects, this disclosure provides a circular RNA prepared by the methods disclosed herein.

[0050] In some respects, this disclosure provides a method for treating a disease or condition in a subject in need, comprising administering the circular RNA disclosed herein to the subject. Attached Figure Description

[0051] Various aspects of this disclosure may take the form of various components and arrangements of components, as well as various steps and arrangements of steps. The accompanying drawings are for illustrative purposes only and should not be considered limiting.

[0052] The features of the embodiments of this disclosure will be more readily understood through the following detailed description taken in conjunction with the accompanying drawings.

[0053] Figure 1A The following is a schematic representation of a genetic construct (DNA or RNA genetic construct): 5'-PHA-3S-E-5S-B-H'-3' in: P contains a promoter; H contains a 5' homologous arm; A contains a 3' intron sequence that contains a catalytic core (CC); 3S contains a 3' splice site; E contains exon sequences; 5S includes a 5' splice site; B contains a 5' intron sequence that contains an internal guide sequence (IGS); H' contains a 3' homologous arm; The promoter (P) should be understood as being included in the DNA genetic construct, but not in the RNA genetic construct.

[0054] Figure 1B The following is a schematic representation of a genetic construct (DNA or RNA genetic construct): 5'-PHA-3S-E-5S-B-H'-3' in: P contains a promoter; H contains a 5' homologous sequence; A contains a 3' intron sequence that contains a catalytic core (CC); 3S contains a 3' splice site; E contains an exon sequence comprising a first excisional scar precursor (ESP) (X) and a second ESP (X'); B contains a 5' intron sequence; and H' contains a 3' homologous arm; The promoter (P) should be understood as being included in the DNA genetic construct, but not in the RNA genetic construct.

[0055] Figure 1C The following is a schematic representation of a genetic construct (DNA or RNA genetic construct): 5'-PA-3S-E-3' in: P contains a promoter; A contains a 3' intron sequence that contains the catalytic core (CC). 3S contains a 3' splice site; and E contains an exon sequence that contains a catalytic nucleotide (CN); The promoter (P) should be understood as being included in the DNA genetic construct, but not in the RNA genetic construct.

[0056] Figure 1D The following is a schematic representation of a genetic construct (DNA or RNA genetic construct): 5'-PA-3S-E-3' in: P contains a promoter; A contains a 3' intron sequence that contains a catalytic core (CC); 3S contains a 3' splice site; E contains an exon sequence that includes: The first part (E') of the exon sequence; First homologous arm (HI) The second part (E'') of this exon sequence The second homologous arm (HI'); and The third exon sequence (E''') contains the catalytic nucleotide (CN); The promoter (P) should be understood as being included in the DNA genetic construct, but not in the RNA genetic construct.

[0057] Figure 1E The following is a schematic representation of a genetic construct (DNA or RNA genetic construct): 5'-PBA-3S-E-3' in: P contains a promoter; B contains a 5' intron sequence that contains an internal guide sequence (IGS); A contains a 3' intron sequence that contains the catalytic core (CC). 3S contains a 3' splice site; E contains an exon sequence that contains a catalytic nucleotide (CN); The promoter (P) should be understood as being included in the DNA genetic construct, but not in the RNA genetic construct.

[0058] Figure 2 This is a schematic diagram of the cyclization mechanism.

[0059] Figure 3A yes Figure 1A The plasmid map of the genetic construct depicted in the image.

[0060] Figure 3B yes Figure 1B The plasmid map of the genetic construct depicted in the image.

[0061] Figure 4 This is a gel analysis of denatured RNA from four group I introns before (-) and after (+) GTP circularization. RNA molecular weight standards are shown on the right side of the figure. The analysis also includes spliced ​​(circularized), unspliced, and nicked RNA samples after GTP, as well as fragment analysis (FA) tailing analysis of multiply RNA.

[0062] Figure 5A This is an analyzer track of a fragment of circularized RNA from an RNA genetic construct in the presence of GTP, containing fragments derived from Anabaena. PCC7120 The sequence of intron sequences.

[0063] Figure 5B It is an analyzer track of a fragment of circularized RNA from an RNA genetic construct in the presence of GTP, which contains a sequence containing intron sequences derived from Anabaena spp.

[0064] Figure 5C It is an analyzer track of a fragment of circularized RNA from an RNA genetic construct in the presence of GTP, which contains a sequence containing an intron sequence derived from the genus *Vibrio zonatiformis*.

[0065] Figure 5D This is an analyzer track of fragments of circularized RNA from an RNA genetic construct in the presence of GTP, which contains fragments derived from... T4 The sequence of introns of bacteriophages.

[0066] Figure 6 This is a flow cytometry analysis of the GFP payload in HEK293T cells transfected with circRNAs derived from different introns indicated by . Detailed Implementation

[0067] This disclosure generally relates to improved genetic constructs for the synthesis of circular RNA. It provides RNA genetic constructs capable of forming circular RNA (i.e., precursor circRNA), DNA genetic constructs from which precursor circRNA can be transcribed, methods for synthesizing circular RNA (i.e., circRNA) using the precursor circRNA, circRNA, and / or DNA genetic constructs disclosed herein, pharmaceutical compositions comprising genetic constructs (e.g., precursor circRNA) and / or cirRNA disclosed herein, and methods for treating a disease in a subject in need of treatment comprising precursor circRNA, circRNA, and / or DNA genetic constructs or pharmaceutical compositions disclosed herein.

[0068] This disclosure includes aspects of synthesizing circRNAs from genetic constructs containing group I introns. The circRNAs discussed in further detail herein may exhibit improved immunogenicity compared to other circRNAs.

[0069] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the event of any conflict, this application (including the definitions) shall prevail. Furthermore, unless the context requires otherwise, singular terms shall include plural terms and plural terms shall include singular terms.

[0070] Throughout this disclosure, the term "an" or "a" entity refers to one or more of the said entities; for example, "a polynucleotide" should be understood to mean one or more polynucleotides. Therefore, the terms "an" (or "a"), "one or more," and "at least one" are used interchangeably herein.

[0071] Furthermore, when used herein, “and / or” will be considered as a specific disclosure of each of the two specified features or components in the presence or absence of the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to include each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0072] It should be understood that wherever the term "comprising" is used to describe an aspect herein, other similar aspects described as "consisting of" and / or "substantially consisting of" are also provided. As used herein, "comprising" is synonymous with "including," "containing," or "characterized in," and is inclusive or open-ended, and does not exclude additional, unlisted elements or method steps. As used herein, "consisting of" excludes any element, step, or component not specified in the elements of the claim. As used herein, "substantially consisting of" does not exclude materials or steps that do not substantially affect the basic and novel features of the claim.

[0073] The term “about” is used herein to mean approximately, roughly, about, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated value. Generally, unless otherwise indicated, the term “about” is used herein to modify numerical values ​​by a variation of around 10% above and below the value (increase or decrease). Unless otherwise indicated, all figures, including the quantities of expressed components, reaction conditions, etc., used in the description of the claims, should be understood to be modified by the term “about” in all cases. Therefore, unless otherwise indicated to the contrary, numerical parameters are approximate values ​​and can vary depending on the desired properties sought to be obtained. At least, and not as an attempt to limit the application of the doctrine of equivalence to the claims, each numerical parameter should be interpreted according to the number of significant figures and ordinary rounding methods.

[0074] The term "at least" preceding a number or series of numbers should be understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that can logically be included, as clearly can be seen from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides in a nucleic acid molecule of 21 nucleotides" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" appears before a series of numbers or ranges, it should be understood that "at least" can modify every number in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18%, regardless of the number of significant figures).

[0075] The terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence,” “nucleic acid sequence,” “polynucleotide,” and their grammatical variations are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine; “RNA molecule”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecule”) or any phosphate ester analogues thereof, such as thiophosphates and thioesters, in single-stranded or double-stranded helical form. A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, particularly DNA or RNA molecules, refers only to the primary and secondary structures of the molecule and is not limited to any particular tertiary form. Therefore, this term includes double-stranded DNA, especially found in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. When discussing the structure of a specific double-stranded DNA molecule, the sequence may be described herein according to the normal convention of giving the sequence only along the non-transcribed DNA strand (i.e., the strand having a sequence homologous to mRNA) in a 5' to 3' orientation. A “recombinant DNA molecule” is a DNA molecule that has undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. The “nucleic acid compositions” of this disclosure comprise one or more nucleic acids as described herein. As described herein, the polynucleotides of this disclosure comprise DNA, RNA, or both. In some respects, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose); polyribonucleotides (containing D-ribose), including tRNA, rRNA, shRNA, siRNA, miRNA, and mRNA, whether spliced ​​or unspliced; any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base; and other polymers containing a positive nucleotide backbone, such as polyamides (e.g., peptide nucleic acid "PNA") and polymorpholinopolymers; and other synthetic sequence-specific nucleic acid polymers, provided that these polymers contain nucleosides in configurations that allow for base pairing and base stacking, such as those found in DNA and RNA.

[0076] It should be understood that when a DNA nucleotide sequence is disclosed herein, the RNA sequence that can be transcribed from it is also considered to be provided herein. Similarly, when an RNA nucleotide sequence is disclosed herein, the DNA sequence that can be transcribed into an RNA sequence is also considered to be provided herein. Thus, for example, when a nucleotide sequence is provided as a DNA sequence (such as AATCGTA), the corresponding RNA sequence AAUCGUA is also considered to be provided; when a nucleotide sequence is provided as an RNA sequence (such as AAUCGUA), the corresponding DNA sequence AATCGTA is also considered to be provided. Those skilled in the art will know how to infer a DNA sequence from a provided RNA sequence (i.e., replacing the U in the provided RNA sequence with T to obtain the corresponding DNA sequence), and how to infer an RNA sequence from a provided DNA sequence (i.e., replacing the T in the provided DNA sequence with U to obtain the corresponding RNA sequence). Those skilled in the art will know which DNA sequences are typically transcribed into RNA and which DNA sequences are typically not transcribed into RNA. For example, those skilled in the art will know that promoter sequences are typically not transcribed from DNA into RNA; while sequences such as exons and introns are typically transcribed from DNA into RNA. Furthermore, those skilled in the art will recognize that some nucleotide sequences, while functionally active only (or primarily) in their DNA form, are also present in the RNA sequences transcribed from them. Similarly, some nucleotide sequences, while functionally active only (or primarily) in their RNA form, are also present in and encoded by the DNA sequence of the transcribed RNA. For example, although RNA molecules are spliced, the nucleotide sequences at the splice sites are also present in and encoded by the DNA sequence of the transcribed RNA.

[0077] The "-" symbol in any formula of the genetic construct disclosed herein can be a phosphodiester bond, or one or more nucleotides inserted between two nucleotides. For example, when the formula requires 5'-AEB-3', where A contains a 3' intron sequence, E contains an exon sequence (i.e., one or more exons or fragments thereof), and B contains a 5' intron sequence, and where the "-" between A and E and / or between E and B can be a phosphodiester bond or one or more nucleotides between A and E and / or between E and B.

[0078] Unless otherwise indicated, as used herein, the term "polypeptide" encompasses both peptides and proteins.

[0079] The term "coding region" refers to a region of DNA or RNA (transcriptional region) that "encodes" a specific protein (such as a payload).

[0080] The term “RNA” is used herein to mean a molecule containing at least one ribonucleotide residue. “Ribonucleotide” refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-furanose group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA (such as partially or completely purified RNA), substantially pure RNA, synthetic RNA, and recombinant RNA (such as modified RNA that differs from naturally occurring RNA by adding, deleting, substituting, and / or altering one or more nucleotides). The term “mRNA” means “messenger RNA” and refers to a “transcript” generated using a DNA template and encoding a peptide or protein. Typically, mRNA contains a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA has only a limited half-life in cells and in vitro. In the context of this disclosure, mRNA can be generated by in vitro transcription from a DNA template. In vitro transcription methods are known to those skilled in the art. For example, various commercially available in vitro transcription kits are available. As further described herein, in some aspects, RNA is linear RNA. In some aspects, RNA is circular RNA. In some aspects, RNA is self-replicating RNA. In some respects, RNA is non-replicating RNA.

[0081] As used herein, the term “precursor circular RNA” refers to RNA (e.g., mRNA) or DNA (also referred to herein as “precursor circRNA”) that is capable of guiding itself to circularize to form circular RNA.

[0082] As used herein, the term "circRNA" or "circRNA" refers to RNA (e.g., mRNA) that forms a circular structure through covalent bonds. circRNA is a single-stranded RNA molecule that lacks free 5' and 3' ends, and therefore lacks a 5' cap and a 3' polyA tail.

[0083] As used herein, the term "payload sequence" refers to a nucleotide sequence that encodes a payload. As used herein, the term "payload" refers to any protein that can be encoded by a payload sequence. In some aspects, payloads include therapeutic proteins. Non-limiting examples of payloads are provided elsewhere in this disclosure.

[0084] The term "sequence identity" is used herein to mean a relationship between two or more amino acid (peptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by sequence comparison. In some respects, sequence identity is calculated based on the full length or a portion thereof of two given SEQ ID NOs. A portion may mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or any other specified percentage, of the two SEQ ID NOs. The term "identity" may also mean sequence correlation between amino acid or nucleic acid sequences, as determined by matching between such sequence strings.

[0085] In some respects, methods for determining identity are designed to give the maximum match between the tested sequences. Methods for determining identity and similarity have been incorporated into publicly available computer programs.

[0086] As used herein, terms such as “effective amount” or “therapeutic effective amount” for circRNA disclosed herein refer to an amount sufficient to achieve a beneficial or desired outcome (including clinical outcome) when administered to a subject (including a human), and thus “effective amount” or its synonyms depend on the context in which they are used.

[0087] As used herein, the term "target cell" refers to a cell in which it is desired to express a payload (e.g., encoded by a payload sequence). As used herein, "non-target cell" refers to a cell in which it is not desired to express a payload.

[0088] As used herein, the term "genetic construct" refers to an artificially designed nucleic acid molecule that can be carried on a vector. A genetic construct can be an RNA nucleic acid molecule (e.g., a precursor circRNA) or a DNA nucleic acid molecule (e.g., an expression cassette that can transcribe precursor circRNA, or a vector containing an expression cassette).

[0089] As used herein, the term "vector" refers to any nucleic acid molecule used for cloning nucleic acids, such as plasmids, bacteriophages, transposons, kinases, chromosomes, artificial chromosomes, viruses, virions, etc. A vector can be a replicon to which another nucleic acid fragment can be ligated to induce replication of the ligated fragment. "Replicon" refers to an autonomous unit that acts as a replicator in vivo, i.e., any genetic element capable of replicating under its own control (e.g., plasmids, bacteriophages, kinases, chromosomes, viruses). A vector can be a "delivery vector," including viral and non-viral vectors for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Vectors can also be used in vitro in the absence of cells to perform any procedure known in the art. For example, a vector can be used for in vitro transcription. A large number of vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. In some aspects, insertion of polynucleotides into a suitable vector can be accomplished by ligating an appropriate polynucleotide fragment into a selected vector having complementary sticky ends. Vectors can be engineered to encode selective markers or reporter genes that provide selection or identification of cells incorporating the vector. The expression of selective markers or reporter genes allows for the identification and / or selection of host cells incorporating and expressing additional coding regions contained on the vector. Examples of selective marker genes known and used in the art include genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, diammonium phosphate herbicide, sulfonamides, etc.; and genes used as phenotypic markers, such as anthocyanin regulatory genes, isopentenyltransferase genes, etc. Examples of reporter genes known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selective markers can also be considered as reporter genes. In some aspects, delivery vectors are selected from the group consisting of: viral vectors (e.g., AAV vectors), plasmids, lipids, protein particles, bacterial vectors, and lysosomes.

[0090] As used in this article, the term "splicing" refers to the process of joining exons while removing introns from the primary transcript (precursor RNA) to form mature RNA.

[0091] An "exon" is a coding segment of a DNA molecule or an RNA molecule transcribed from a DNA molecule and then translated into a protein, or part of mature structural RNA (e.g., tRNA or rRNA). In most eukaryotic genes, exons are separated by "introns," which are inserted into non-coding segments of DNA. Therefore, introns are nucleic acid segments transcribed into RNA but are removed by splicing and are thus not present in mature RNA transcripts containing only exons. Before splicing occurs, a new, immature strand of messenger or structural RNA is called precursor RNA and can contain both introns and exons; the RNA molecule produced by splicing is called RNA or mature RNA. The precursor RNA molecule undergoes splicing, and only the exons are retained in the "mature RNA," which is then translated into protein in the case of mRNA. "Structural RNA" or "non-coding RNA" is an RNA molecule that does not translate into protein and functions as an RNA molecule. Exemplary structural RNA molecules are rRNA and tRNA. During splicing, introns are removed from the precursor RNA by cutting at a conserved sequence called the "splicing site," which is located at the 5' and 3' ends of the exon.

[0092] As used herein, the term "exon sequence" refers to the nucleotide sequence included in the mature RNA after splicing. As used herein, the term "intron sequence" refers to the nucleotide sequence removed from the RNA molecule during splicing and therefore not included in the mature RNA after splicing. An exon sequence may contain one or more exons or portions thereof, may contain nucleotide sequences derived from naturally occurring nucleotide sequences, or may be an artificial sequence. An intron sequence may contain one or more introns or portions thereof, may contain nucleotide sequences derived from naturally occurring nucleotide sequences, or may be an artificial sequence.

[0093] As used herein, the terms "5' intron sequence" and "3' intron sequence" refer to intron sequences located at the 5' and 3' of the intron sequence, respectively. In some aspects, the constructs disclosed herein contain "5' intron sequences" and "3' intron sequences" in opposite positions. Thus, in some aspects, a "3' intron sequence" may be located at the 5' of the constructs disclosed herein, and a "5' intron sequence" may be located at the 3' of the constructs disclosed herein.

[0094] As used herein, the term "splicing site" refers to the sequence at the boundary between an exon and an intron. A splicing site contains a pair of nucleotides linked by a phosphodiester bond, which is cleaved during the splicing reaction.

[0095] The 5' splice site is located at the boundary between the 3' end of an exon and the 5' end of an intron. The 5' splice site contains a pair of nucleotides, between which a phosphodiester bond is cleaved during the splicing reaction. The nucleotide at the 5' end of this pair is contained within the exon, and the nucleotide at the 3' end is contained within the intron.

[0096] The 3' splice site is located at the boundary between the 5' end of an exon and the 3' end of an intron. The 3' splice site contains a pair of nucleotides, between which a phosphodiester bond is cleaved during the splicing reaction. The nucleotide at the 5' end of this pair is contained within the intron, and the nucleotide at the 3' end is contained within the exon.

[0097] As used herein, the term "catalytic nucleotide" refers to a nucleotide that promotes the circocyclization of precursor circRNA through interaction with the catalytic core of an intron sequence.

[0098] A “linear nucleic acid molecule” is described as having a “5’-terminus” and a “3’-terminus” because the phosphodiester bond linkage of a nucleic acid occurs at the 5’ and 3’ carbons of the sugar moiety of the substituent mononucleotide. The terminal nucleotide of a polynucleotide that will form a new link with the 5’ carbon is its 5’-terminal nucleotide. The terminal nucleotide of a polynucleotide that will form a new link with the 3’ carbon is its 3’-terminal nucleotide. As used herein, a terminal nucleotide is the nucleotide at the 3’- or 5’-terminal position.

[0099] "Transcription" refers to the process of forming or synthesizing RNA molecules using DNA molecules as templates via RNA polymerase. This invention does not limit the RNA polymerase used for transcription. For example, in some embodiments, T7 RNA polymerase may be used.

[0100] "Translation" refers to the formation of polypeptide molecules based on RNA templates via ribosomes.

[0101] As used herein, the terms “coding sequence” or “expression sequence” refer to the nucleic acid sequence (i.e., protein-coding sequence) that encodes a product (e.g., a peptide or polypeptide), or a regulatory or structural nucleic acid (i.e., tRNA or rRNA).

[0102] As used herein, a "spacer region" refers to a region of a polynucleotide sequence, ranging from one nucleotide to hundreds or thousands of nucleotides, that separates two other elements along the polynucleotide sequence. The sequence can be defined or it can be random. Spacer regions are typically non-coding. In some embodiments, spacer regions include double-strand-forming regions.

[0103] As used herein, "internal ribosome entry site" or "IRES" refers to an RNA sequence or structural element ranging in size from 10 nt to 1000 nt or larger that can initiate polypeptide translation in the absence of a typical RNA cap structure. IRES are typically about 500 nt to about 700 nt in length.

[0104] II. Precursor circular RNA and DNA genetic constructs This document provides RNA or DNA genetic constructs for the synthesis of circular RNA (circRNA). This disclosure also provides circRNAs generated from such genetic constructs. In some aspects, the genetic constructs for the synthesis of circRNA are RNA genetic constructs (i.e., precursor circRNAs). In some aspects, the genetic constructs for the synthesis of circRNA are DNA genetic constructs, from which transcription is possible. In some aspects, the genetic constructs disclosed herein, or genetic constructs transcribed from the DNA genetic constructs disclosed herein, undergo a splicing reaction. In some aspects, the splicing reaction is an autocatalytic splicing reaction (i.e., a self-splicing reaction).

[0105] In some respects, the genetic constructs disclosed herein comprise one or more intron sequences (i.e., one or more introns or fragments thereof), one or more exon sequences (i.e., one or more exons or fragments thereof), and one or more splicing sites.

[0106] In some respects, the genetic constructs disclosed herein comprise one or more intron sequences (i.e., one or more introns or fragments thereof), one or more exon sequences (i.e., one or more exons or fragments thereof), and one or more catalytic nucleotides.

[0107] In some respects, the genetic constructs disclosed herein comprise one or more intron sequences (i.e., one or more introns or fragments thereof), one or more exon sequences (i.e., one or more exons or fragments thereof), one or more splice sites, and one or more catalytic nucleotides.

[0108] In some respects, the genetic constructs disclosed herein are DNA genetic constructs (e.g., contained in a vector).

[0109] In some aspects, the genetic constructs disclosed herein are RNA genetic constructs. In some aspects, the RNA genetic constructs disclosed herein undergo a splicing reaction. In some aspects, the splicing reaction is an autocatalytic splicing reaction. In some aspects, the splicing reaction partially removes one or more intron sequences (i.e., one or more introns or fragments thereof) from the RNA genetic constructs disclosed herein. In some aspects, the splicing reaction completely removes one or more intron sequences (i.e., one or more introns or fragments thereof) from the RNA genetic constructs disclosed herein. In some aspects, the splicing reaction circularizes one or more exon sequences (i.e., one or more exons or fragments thereof) contained in the RNA genetic constructs disclosed herein.

[0110] In some respects, one or more circularized exon sequences (i.e., one or more exons or fragments thereof) are not protein-coding exon sequences. In some respects, one or more circularized exon sequences (i.e., one or more exons or fragments thereof) are structural RNA. In these respects, one or more circularized exon sequences (i.e., one or more exons or fragments thereof) can be therapeutic agents (i.e., therapeutic RNA). In some respects, one or more exon sequences (i.e., one or more exons or fragments thereof) are protein-coding sequences. In these respects, one or more exon sequences (i.e., one or more exons or fragments thereof) can encode therapeutic agents (i.e., therapeutic proteins). In these respects, one or more exon sequences (i.e., one or more exons or fragments thereof) are translated into therapeutic agents (i.e., therapeutic proteins).

[0111] In some aspects, the genetic construct is a linear RNA produced by in vivo transcription of the DNA genetic construct disclosed herein. In some aspects, the genetic construct (e.g., a precursor circRNA) is a linear RNA produced by in vitro transcription of the DNA genetic construct disclosed herein. In some aspects, the genetic construct (e.g., a precursor circRNA) is a linear RNA produced by chemical synthesis. The genetic constructs disclosed herein (e.g., precursor circRNA) may be unmodified, partially modified, or fully modified.

[0112] In some aspects, the genetic constructs disclosed herein (e.g., precursor circRNAs) can be circularized to produce circular RNA (circRNA). In some aspects, the circRNAs disclosed herein comprise exon sequences (i.e., one or more exons) contained within the DNA or RNA genetic constructs disclosed herein. In some aspects, the circRNAs disclosed herein consist of exon sequences (i.e., one or more exons) contained within the DNA or RNA genetic constructs disclosed herein. In some aspects, the circRNAs disclosed herein also comprise one or more nucleotides contained within the DNA or RNA genetic constructs disclosed herein that are not part of an exon sequence (i.e., one or more exons). One or more nucleotides contained within the DNA or genetic constructs disclosed herein that are not part of an exon sequence (i.e., one or more exons) may, for example, be contained in intron sequences, splice sites, homologous arms of the DNA genetic construct, or the genetic constructs disclosed herein, or any combination thereof. In these respects, the circRNAs disclosed herein are described as containing excision scars. In some respects, the size of the circular RNA is at least 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, or 4500 nucleotides. The circRNAs disclosed herein may be unmodified, partially modified, or fully modified.

[0113] In some aspects, the RNA or DNA genetic constructs capable of forming the circular RNAs disclosed herein (i.e., precursor circRNAs or DNA genetic constructs capable of transcribing precursor circRNAs) comprise one or more intron sequences (i.e., one or more introns or fragments thereof) and / or one or more exon sequences (i.e., one or more exons or fragments thereof). In some aspects, the one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from the nucleotide sequences of group I introns.

[0114] IIA. Genetic constructs IIA1. Genetic construct A.

[0115] This disclosure provides a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA), comprising the formula 5'-HAEB-H'-3', wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence (i.e., one or more exons or fragments thereof), B contains a 5' intron sequence, and H' contains a 3' homologous arm. In some aspects, the construct contains one or more splicing sites, for example, between A and E and / or between E and B. In some aspects, the 3' end of A and the 5' end of E form a 3' splicing site (3S), and the 3' end of E and the 5' end of B form a 5' splicing site (5S). In some aspects, the construct also contains a promoter, wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct. In some aspects, the 5' and 3' intron sequences are reverse complementary to each other.

[0116] In some aspects, this disclosure provides a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA), comprising the formula 5'-PHA-3S-E-5S-B-H'-3', wherein: P contains a promoter (e.g., T7, T3, SP6, etc.), H contains a 5' homologous arm, A contains a 3' intron sequence, 3S contains a 3' splice site, E contains an exon sequence (i.e., one or more exons or fragments thereof), 5S contains a 5' splice site, B contains a 5' intron sequence, H' contains a 3' homologous arm, and wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct. In some aspects, the 5' and 3' intron sequences are inversely complementary to each other. In some aspects, the 3' end of A and the 5' end of E form a 3' splice site (3S), and the 3' end of E and the 5' end of B form a 5' splice site (5S).

[0117] This disclosure also relates to a method for generating circular RNA, comprising adding GTP to a genetic construct (e.g., DNA or RNA) having a nucleotide sequence of the formula 5'-HAEB-H'-3', wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence (i.e., one or more exons or fragments thereof), B contains a 5' intron sequence, and H' contains a 3' homologous arm or the formula 5'-PHA-3S-E-5S-B-H'-3', wherein: P contains a promoter (e.g., T7, T3, SP6, etc.), H contains a 5' homologous arm, A contains a 3' intron sequence, 3S contains a 3' splice site, E contains an exon sequence (i.e., one or more exons or fragments thereof), 5S contains a 5' splice site, B contains a 5' intron sequence, and H' contains a 3' homologous arm, and wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct. In some respects, the 3' end of A and the 5' end of E form a 3' splice site (3S), and the 3' end of E and the 5' end of B form a 5' splice site (5S).

[0118] In some respects, this disclosure includes a circular RNA which is produced by a precursor circRNA or method described herein.

[0119] IIA2. Genetic construct B.

[0120] This disclosure also provides a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA), comprising the formula: 5'-HAEB-H'-3', wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence comprising a first resection scar precursor (X) and a second resection scar precursor X', B contains a 5' intron sequence, and H' contains a 3' homologous arm. In some aspects, the construct further comprises one or more splicing sites, for example, between A and E (3S) and / or between E and B (5S). In some aspects, the 3' end of A and the 5' end of E form a 3' splicing site (3S), and the 3' end of E and the 5' end of B form a 5' splicing site (5S). In some aspects, the construct further comprises a promoter, wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct. In some respects, the 5' and 3' intron sequences are reverse complementary to each other.

[0121] In some aspects, this disclosure also provides a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA), comprising the following formula: 5'-PHA-3S-E-5S-B-H'-3', wherein: P contains a promoter (e.g., T7, T3, SP6, etc.), H contains a 5' homologous arm, A contains a 3' intron sequence, 3S contains a 3' splice site, E contains an exon sequence comprising a first resection scar precursor (X) and a second resection scar precursor X', 5S contains a 5' splice site, B contains a 5' intron sequence, and H' contains a 3' homologous arm; and wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct. In some aspects, the 5' and 3' intron sequences are reverse complementary to each other. In some respects, the 3' end of A and the 5' end of E form a 3' splice site (3S), and the 3' end of E and the 5' end of B form a 5' splice site (5S).

[0122] This disclosure also relates to a method for generating circular RNA, comprising adding GTP to a genetic construct (e.g., DNA or RNA) containing a nucleotide sequence having the formula 5'-HAEB-H'-3', wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence comprising a first excisional scar precursor (X) and a second excisional scar precursor X', B contains a 5' intron sequence, and H' contains a 3' homologous arm or the following formula: 5'-PHA-3S-E-5S-B -H'-3', where: P contains a promoter (e.g., T7, T3, SP6, etc.), H contains a 5' homologous arm, A contains a 3' intron sequence, 3S contains a 3' splice site, E contains an exon sequence containing a first excisional scar precursor (X) and a second excisional scar precursor X', 5S contains a 5' splice site, B contains a 5' intron sequence, and H' contains a 3' homologous arm; and the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct. In some respects, the 3' end of A and the 5' end of E form a 3' splice site (3S), and the 3' end of E and the 5' end of B form a 5' splice site (5S).

[0123] In some respects, this disclosure includes a circular RNA which is produced by a precursor circRNA or method described herein.

[0124] IIA3. Genetic construct C.

[0125] This disclosure provides a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA), comprising the formula: 5'-AE-3', where A contains a 3' intron sequence and E contains an exon sequence containing a catalytic nucleotide (CN). In some aspects, the construct also contains a splicing site, for example, between A and E (3S). In some aspects, the construct also contains a promoter, wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct. In some aspects, the 5' and 3' intron sequences are reverse complementary to each other.

[0126] This disclosure provides a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA), comprising the following formula: 5'-PA-3S-E-3', wherein P contains a promoter (e.g., T7, T3, SP6, etc.), A contains a 3' intron sequence, 3S contains a 3' splice site, and E contains an exon sequence containing a catalytic nucleotide (CN); and wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct.

[0127] This disclosure also relates to a method for generating circular RNA, comprising allowing a genetic construct (e.g., DNA or RNA) to self-circularize, wherein the genetic construct comprises a nucleotide sequence having the following formula: 5'-AE-3', where A contains a 3' intron sequence and E contains an exon sequence containing a catalytic nucleotide (CN), or a nucleotide sequence having the following formula: 5'-PA-3S-E-3', where P contains a promoter (e.g., T7, T3, SP6, etc.), A contains a 3' intron sequence, 3S contains a 3' splice site, and E contains an exon sequence containing a catalytic nucleotide (CN); and wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct.

[0128] In some respects, this disclosure includes a circular RNA which is produced by a precursor circRNA or method described herein.

[0129] IIA4. Genetic construct D.

[0130] This disclosure provides a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA), comprising the formula: 5'-AE-3', where A contains a 3' intron sequence comprising a catalytic core (CC); E contains an exon sequence comprising: (i) a first portion of the exon sequence (E'); (ii) a first homologous arm (HI); (iii) a second portion of the exon sequence (E''); (iv) a second homologous arm (HI'); and (v) a third exon sequence comprising a catalytic nucleotide (CN) (E'''). In some aspects, the construct also includes a splicing site, for example, between A and E (3S), for example, at the 3' end of A. In some aspects, the construct also includes a promoter, wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct. In some aspects, the 5' and 3' intron sequences are reverse complementary to each other. In some respects, the 3' end of A and the 5' end of E form a 3' splice site (3S).

[0131] In some aspects, this disclosure provides a genetic construct comprising a nucleotide sequence having the following formula: 5'-PA-3S-E-3', wherein P is a promoter (e.g., T7, T3, SP6, etc.), A comprises a 3' intron sequence containing a catalytic core (CC); 3S comprises a 3' splice site; E comprises an exon sequence containing: (i) a first portion of the exon sequence (E'); (ii) a first homologous arm (HI); (iii) a second portion of the exon sequence (E''); (iv) a second homologous arm (HI'); and (v) a third exon sequence containing a catalytic nucleotide (CN) (E'''), and wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct.

[0132] This disclosure also relates to a method for generating circular RNA, comprising allowing a genetic construct (e.g., DNA or RNA) to self-circularize, wherein the genetic construct comprises a nucleotide sequence having the following formula: 5'-AE-3', where A comprises a 3' intron sequence containing a catalytic core (CC); E comprises an exon sequence containing: (i) a first portion of the exon sequence (E'); (ii) a first homologous arm (HI); (iii) a second portion of the exon sequence (E''); (iv) a second homologous arm (HI'); and (v) a third exon sequence containing a catalytic nucleotide (CN) (E'''), or a nucleotide sequence having the following formula: 5'-PA-3S-E-3', where P is a promoter (e.g., T7, T3, SP6, etc.), A contains a 3' intron sequence containing a catalytic core (CC); 3S contains a 3' splice site; E contains an exon sequence containing: (i) a first portion of the exon sequence (E'); (ii) a first homologous arm (HI); (iii) a second portion of the exon sequence (E''); (iv) a second homologous arm (HI'); and (v) a third exon sequence containing a catalytic nucleotide (CN) (E'''), and wherein the promoter (P) should be understood as being included in the DNA genetic construct but not in the RNA genetic construct. In some respects, the 3' end of A and the 5' end of E form a 3' splice site (3S).

[0133] In some respects, this disclosure includes a circular RNA which is produced by a precursor circRNA or method described herein.

[0134] IIA5. Genetic construct E.

[0135] This disclosure provides a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA), comprising the formula: 5'-BAE-3', wherein B contains a 5' intron sequence comprising an internal guide sequence (IGS), A contains a 3' intron sequence, and E contains an exon sequence comprising a catalytic nucleotide. In some aspects, the construct includes a splicing site between A and E. In some aspects, the 3' end of A and the 5' end of E form a 3' splicing site (3S). In some aspects, the construct includes a promoter, wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct.

[0136] This disclosure provides a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA), comprising the following formula: 5'-PBA-3S-E-3', wherein P contains a promoter (e.g., T7, T3, SP6, etc.), B contains a 5' intron sequence containing an internal guide sequence (IGS), A contains a 3' intron sequence, 3S contains a 3' splicing site, and E contains an exon sequence containing a catalytic nucleotide; and wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct.

[0137] This disclosure also relates to a method for generating circular RNA, comprising allowing a genetic construct (e.g., DNA or RNA) to self-circulate, wherein the genetic construct comprises a nucleotide sequence having the following formula: 5'-BAE-3', wherein B comprises a 5' intron sequence containing an internal guide sequence (IGS), A comprises a 3' intron sequence, and E comprises an exon sequence containing a catalytic nucleotide, or a nucleotide sequence having the following formula: 5'-PBA-3S-E-3', wherein P comprises a promoter (e.g., T7, T3, SP6, etc.), B comprises a 5' intron sequence containing an internal guide sequence (IGS), A comprises a 3' intron sequence, 3S comprises a 3' splice site, and E comprises an exon sequence containing a catalytic nucleotide; and wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct.

[0138] In some respects, this disclosure includes a circular RNA which is produced by a precursor circRNA or method described herein.

[0139] II.B. Components of Genetic Constructs II.B.1. Splice Site In some respects, genetic constructs capable of forming the circular RNAs disclosed herein (i.e., precursor circRNAs or DNA genetic constructs capable of transcribing precursor circRNAs) contain or form 5' splice sites, 3' splice sites, catalytic nucleotides, or combinations thereof.

[0140] In some respects, genetic constructs capable of forming the circular RNAs disclosed herein (i.e., precursor circRNAs or DNA genetic constructs capable of transcribing precursor circRNAs) contain 5' splice sites and 3' splice sites.

[0141] In some respects, genetic constructs capable of forming the circular RNAs disclosed herein (i.e., precursor circRNAs or DNA genetic constructs capable of transcribing precursor circRNAs) contain 3' splicing sites and catalytic nucleotides.

[0142] In some respects, the 5' and 3' splice sites are interchanged relative to their original positions in the naturally occurring configuration. In the naturally occurring configuration, the 5' splice site is upstream of the exon, and the 3' splice site is downstream of the exon sequence. In the genetic construct disclosed herein, the 5' splice site may be downstream of the exon sequence, and the 3' splice site may be upstream of the exon sequence; that is, the 5' and 3' splice sites are interchanged relative to their original positions in the naturally occurring configuration.

[0143] In some respects, the 3' splice site is located upstream of the exon sequence, and the catalytic nucleotide is located downstream of the exon sequence.

[0144] A splice site is a nucleotide sequence located at the boundary between an intronic sequence and an exon sequence, and contains a pair of nucleotides linked by a phosphodiester bond that is cleaved during the splicing reaction. Thus, each splice site contains a portion within the intronic sequence and a portion within the exon sequence. In some respects, a splice site is a 5' splice site. A 5' splice site is located at the boundary between the 3' end of the exon sequence and the 5' end of the intron sequence. A 5' splice site contains a pair of nucleotides between which a phosphodiester bond is cleaved during the splicing reaction. The nucleotide at the 5' of this pair (also referred to herein as "nucleotide at the 5' of the 5' splice site" or "5S(5')") is contained within the exon sequence, and the nucleotide at the 3' of this pair (also referred herein as "nucleotide at the 3' of the 5' splice site" or "5S(3')") is contained within the intron sequence.

[0145] In some respects, 5S(5') is U or T, and 5S(3') is N, where N is any one of A, C, G or U.

[0146] In some respects, the 5' splice site comprises a sequence of the formula 5'-U (or T)-N-3', wherein the splice site is between U (or T) and N, and wherein N is any one of A, C, G, T or U.

[0147] In some respects, the splice site is a 3' splice site. A 3' splice site is located at the boundary between the 5' end of the exon sequence and the 3' end of the intron sequence. A 3' splice site contains a pair of nucleotides between which a phosphodiester bond is cleaved during the splicing reaction. The nucleotide at the 5' of this pair (also referred to herein as the "nucleotide at the 5' of the 3' splice site" or "3S(5')") is contained within the intron sequence, and the nucleotide at the 3' of this pair (also referred to herein as the "nucleotide at the 3' of the 53' splice site" or "3S(3')") is contained within the exon sequence.

[0148] In some respects, 3S(5') is G and 3S(3') is N, where N is any one of A, C, G, T or U.

[0149] In some respects, the 3' splice site comprises a sequence of the formula 5'-GN-3', wherein the splice site is between G and N, and wherein N is any one of A, C, G, T or U.

[0150] In some respects, genetic constructs capable of forming the circular RNAs disclosed herein (i.e., precursor circRNAs or DNA genetic constructs capable of transcribing precursor circRNAs) contain 5' splice sites and 3' splices.

[0151] In some respects, genetic constructs capable of forming the circular RNAs disclosed herein (i.e., precursor circRNAs or DNA genetic constructs capable of transcribing precursor circRNAs) contain splicing sites and catalytic nucleotides.

[0152] In some respects, genetic constructs capable of forming the circular RNAs disclosed herein (i.e., precursor circRNAs or DNA genetic constructs capable of transcribing precursor circRNAs) contain 3' splicing sites and catalytic nucleotides.

[0153] In some aspects, the 3' splice site comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequence of SEQ ID NO: 31-36. In some aspects, the 3' splice site comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequence of SEQ ID NO: 33. In some aspects, the 3' splice site is composed of the nucleotide sequence of SEQ ID NO: 31-36. In some aspects, the 3' splice site is composed of the nucleotide sequence of SEQ ID NO: 33.

[0154] In some aspects, the 5' splice site comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequence of SEQ ID NO: 37-41 or the nucleotide sequence of NAGU^AAA, NCUU^, NAGU^, NCUU^, or NGGU^, wherein N is A, U, C, or G, and wherein “^” indicates the position of the phosphodiester bond cleaved during the splicing reaction. In some aspects, the 5' splice site comprises the nucleotide sequence of NAGU^AAA, wherein N is A, U, C, or G, and wherein “^” indicates the position of the phosphodiester bond cleaved during the splicing reaction. In some aspects, the 5' splice site consists of the nucleotide sequence of SEQ ID NO: 37-41 or the nucleotide sequence of NAGU^AAA, NCUU^, NAGU^, NCUU^, or NGGU^, where N is A, U, C, or G, and where “^” indicates the position of the phosphodiester bond cleaved during the splicing reaction. In some aspects, the 5' splice site consists of the nucleotide sequence of NAGU^AAA, where N is A, U, C, or G, and where “^” indicates the position of the phosphodiester bond cleaved during the splicing reaction. Non-limiting examples of 5' and 3' splice sites that can be used in this genetic construct are shown in Table 1.

[0155] Table 1. Exemplary splice sites (“^” indicates the location of the phosphodiester bond that is cleaved during the splicing reaction).

[0156] II.B.2 Introns Splicing is the process of converting precursor RNA transcripts (i.e., primary transcripts) into mature RNA molecules (e.g., mature mRNA, tRNA, or rRNA). During splicing, introns are removed from the precursor RNA molecule (i.e., primary transcript) and exons are joined together. For many eukaryotic RNAs, splicing occurs in a series of reactions catalyzed by the spliceosome, a complex of small nucleoribonucleoproteins (snRNPs), but self-splicing introns can catalyze their own excision from their parent RNA molecule. For example, group I introns can catalyze their own excision from their parent RNA molecule without the aid of accessory proteins.

[0157] Group I introns are self-splicing introns that can catalyze the excision of themselves from primary transcripts (e.g., pre-mRNA, pre-tRNA, and pre-rRNA) in a wide range of organisms. The self-splicing ability of Group I introns enables them to function as ribozymes.

[0158] Group I introns are found in the genomes of bacteria, bacteriophages, viruses, and organelles, and are also commonly found in the nuclear rDNA genes of fungi, plants, and algae.

[0159] Group I introns are highly variable at the primary sequence level but possess characteristically conserved secondary and tertiary structures. Structural studies revealed a well-defined and highly conserved RNA core responsible for catalysis, although universally conserved nucleotide residues are almost absent in Group I introns. The secondary structure of Group I introns consists of paired (P) elements designated P1 through P13 and a single-stranded loop region. Short conserved sequences can be identified in some intron sequences. The P1 and P10 helices form the substrate-binding domain, with the 5' and 3' splice sites juxtaposed. P2 is absent in some Group I introns. The active core of Group I ribozymes is assembled from two helical domains: P4 / P6 (P4, P5, and P6), which are considered scaffold domains, and P3 / P9 (P3, P7, P8, and P9), which form the catalytic domain. The P3-P7-P9 helix contains a guanosine-5'-triphosphate (GTP) binding pocket, and exogenous GTP docks at the G-binding site located in P7. Here, the 3'–OH of the exogenous GTP is positioned such that it can attack the 5'–3' phosphodiester bond located at the 5' splice site within the P1 fold. In some respects, P10 contains the nucleotide sequence TAAGGTA (or UAGGUA) or CT (or CU).

[0160] Group I introns are removed from precursor RNA via an autocatalytic RNA splicing event mediated by the intron's RNA tertiary structure. Base-pairing interactions between the 5' end of the intron and flanking exon sequences define the locations of the 5' and 3' splice sites. The internal guide sequence (IGS), a short intron sequence near the 5' end, pairs with the upstream exon sequence to form P1, determining the 5' splice site. The 3' splice site is determined by pairing a short downstream exon sequence with a portion of the IGS to form P10, mediating the interaction between P9 and the P3 / P8 helix that forms the catalytic core.

[0161] Splicing of group I intron RNAs is achieved through a two-step transesterification reaction with exogenous GTP (αG), whose 3'–OH acts as the initiating nucleophile. Binding of αG to the G-binding site in P7 positions the 3'–OH of GTP to attack the 5' splice site. During the first transesterification step, αG is attached to the 5' end of the intron RNA via a 3'-5' phosphodiester bond. This step is followed by a conformational change that allows the terminal 3' guanosine (ωG) of the upstream exon to exchange position with αG and occupy the G-binding site, initiating the second transesterification reaction. The 3'–OH of the upstream exon attacks the 3' splice site (an interaction facilitated by the formation of P10), promoting the ligation of the upstream and downstream exons and the release of the intron RNA. A non-limiting example of P10 is shown below: Table 2.

[0162] In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from a single intron. In other respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from two or more different introns.

[0163] In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are artificial sequences containing nucleotide sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences of naturally occurring introns. In some respects, introns are group I.

[0164] In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from introns derived from bacterial genomes, bacteriophage genomes, viral genomes, eukaryotic organelle genomes, or nuclear genomes of fungi, plants, or algae, or RNA transcribed from them. In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from bacteriophage genomes or RNA transcribed from them. In some respects, intron sequences are derived from the T4 bacteriophage genome or RNA transcribed from it. In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from bacterial genomes or RNA transcribed from them. In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from cyanobacterial genomes or RNA transcribed from them. In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from *Azolla oblongata*, *Azolla oblongata*, or *Azolla oblongata*. PCC7120The genome of *Strombyx mori* (e.g., strain NIVA-CYA142) or *Synechococcus* may be derived from or derived from RNA transcribed from them. In some aspects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from the genome of nitrogen-fixing bacteria or from RNA transcribed from them. In some aspects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from the genome of *Vibrio azooptera* or from RNA transcribed from them. In some aspects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from the genome of protozoa or from RNA transcribed from them. In some aspects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from the genome of ciliates or from RNA transcribed from them. In some aspects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from the genome of *Tetrahymena thermophila* or from RNA transcribed from them. In some aspects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from the genome of fungi or from RNA transcribed from them. In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from the genus *Trimorphomyces*. Trimorphomyces The genome or RNA derived from its transcription. In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from the genome of *Papilionella* or from its transcribed RNA.

[0165] In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from genomic regions of protein-coding RNA or structural RNA (e.g., tRNA, rRNA) or from RNA transcribed from them.

[0166] In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from introns of thymidine synthase (td) from T4 phage; derived from *Azolla oblongata* and *Azolla oblongata*. PCC7120 Precursor tRNAs from *Strombyx mori* (e.g., strain NIVA-CYA 142) or *Synechococcus*. LEU Introns; precursor tRNA derived from *Vibrio zonesia* ILE Introns; introns derived from the 26S ribosomal RNA of Tetrahymena thermophila; cytochrome C oxidase subunit 2 derived from Papilionophora fungi (… COX2 Introns of , or any combination thereof. In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from introns contained in the GISSD database (http: / / www.rna.whu.edu.cn / gissd / ).

[0167] In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from introns of thymidine synthase (td) from T4 phage.

[0168] In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from precursor tRNAs from *Anabaena spp.* LEU Introns.

[0169] In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from Anabaena. PCC7120 Pre-tRNA LEU Introns.

[0170] In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from precursor tRNAs from *Hylocereus algae* (e.g., strain NIVA-CYA 142). LEU Introns.

[0171] In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from precursor tRNAs from Synechococcus. LEU Introns.

[0172] In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from precursor tRNAs of the genus *Vibrio azotocinus*. ILE Introns.

[0173] In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from introns of 26S ribosomal RNA from Tetrahymena thermophila.

[0174] In some respects, one or more intron sequences (i.e., one or more introns or fragments thereof) are derived from the cytochrome C oxidase subunit 2 of *Papilionella*. COX2 Introns of ).

[0175] In some aspects, one or more intron sequences (i.e., one or more introns or fragments thereof) comprise nucleotide sequences derived from introns having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of SEQ ID NO: 1-5, 6-10, 139-141, or 142-144. In some aspects, one or more intron sequences (i.e., one or more introns or fragments thereof) comprise nucleotide sequences derived from introns having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of SEQ ID NO: 3 or 8. In some aspects, one or more intron sequences (i.e., one or more introns or fragments thereof) comprise nucleotide sequences derived from introns composed of any of SEQ ID NO: 1-5, 6-10, 139-141, or 142-144. In some aspects, one or more intron sequences (i.e., one or more introns or fragments thereof) comprise nucleotide sequences derived from introns composed of SEQ ID NO: 3 or 8.

[0176] Non-restrictive examples of introns that can be used in this genetic construct are shown in Table 2.

[0177] Table 3. Exemplary Introns

[0178] When referring to precursor circRNA, "introns derived from" means intron sequences containing intron sequences derived from RNA encoded by a gene. When referring to DNA genetic constructs (from which precursor circRNA can be transcribed), "introns derived from" means intron sequences containing DNA sequences derived from a gene.

[0179] In some aspects, the intron sequences (i.e., one or more introns or fragments thereof) contained in a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA) contain the nucleotide sequences of a full-length intron. In some aspects, the intron sequences (i.e., one or more introns or fragments thereof) contained in a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA) do not contain the nucleotide sequences of a full-length intron.

[0180] In some respects, the intron sequence contains less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, and less than 1%.

[0181] In some aspects, the intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of SEQ ID NO: 1-5, 6-10, 139-141, or 142-144. In some aspects, the intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of SEQ ID NO: 3 or 8. In some aspects, the intron sequence consists of a nucleotide sequence of any of SEQ ID NO: 1-5, 6-10, 139-141, or 142-144. In some aspects, the intron sequence consists of a nucleotide sequence of SEQ ID NO: 3 or 8.

[0182] In some respects, the intron sequence (i.e., one or more introns or fragments thereof) contained in a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA) is split into two segments.

[0183] In some respects, the intron sequence is divided into 5' segments and 3' segments. The 5' segment of the intron sequence is also referred to herein as the "5' intron sequence", and the 3' segment of the intron sequence is also referred to herein as the "3' intron sequence".

[0184] It should be understood that the 5' and 3' descriptors of the intron fragments refer only to the positions of the two intron fragments relative to the full-length sequence of the intron, and both the 5' and 3' intron sequences can contain any portion of the intron sequence, as long as the intron sequence required for circularization (e.g., IGS, ωG, or catalytic core) is present in the intron sequence. For example, an intron sequence contained in a genetic construct disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA) may contain a sequence comprising 80% of the full-length intron sequence (i.e., an intron fragment comprising 80% of the full-length intron sequence), wherein the full-length intron has a length of 1000 nucleotides (nucleotides 1000 from nucleotide 1 at 5' to nucleotide 1000 at 3'). Such an exemplary intron sequence contains 800 nucleotides. The 800 nucleotides can correspond, for example, to nucleotides 1-300 and 501-1000 of the 1000 nucleotides of a full-length intron, i.e., nucleotides 1 to 300 and 501 to 1000 of the full-length intron. This exemplary intron sequence does not contain nucleotides 301-500 of the full-length intron. In this exemplary case, the 5' intron sequence can contain, for example, 300 nucleotides that correspond to nucleotides 1-300 of the 1000 nucleotides of a full-length intron, and the 3' intron sequence can contain, for example, 500 nucleotides that correspond to nucleotides 501-1000 of the 1000 nucleotides of a full-length intron.

[0185] Furthermore, both the 5' and 3' intron sequences can contain nucleotide sequences that include discontinuous nucleotides within the full-length intron sequence. For example, an exemplary intron sequence comprising 80% of the full-length intron sequence (i.e., an intron fragment comprising 80% of the full-length intron sequence) contains 800 nucleotides, where the full-length intron has a length of 1000 nucleotides (from nucleotide 1 at 5' to nucleotide 1000 at 3'). In this exemplary case, the 5' intron sequence may contain, for example, 200 nucleotides that correspond to nucleotides 1-50 and 100-250 of the 1000 nucleotides of the full-length intron, i.e., nucleotides 1 to nucleotide 50 and nucleotides 100 to nucleotide 250 of the full-length intron, and the 3' intron sequence may contain, for example, 600 nucleotides that correspond to nucleotides 401-1000 of the 1000 nucleotides of the full-length intron.

[0186] In some respects, the genetic constructs capable of forming the circular RNAs disclosed herein (i.e., precursor circRNAs or DNA genetic constructs capable of transcribing precursor circRNAs) contain 5' intron sequences and 3' intron sequences. In these respects, the 5' intron sequences and 3' intron sequences are included in cis in the same genetic construct.

[0187] In some aspects, the genetic constructs capable of forming the circular RNAs disclosed herein (i.e., precursor circRNAs or DNA genetic constructs capable of transcribing precursor circRNAs) contain 5' intron sequences and do not contain 3' intron sequences.

[0188] In some aspects, the 5' intron sequence and the 3' intron sequence are derived from the same intron. In some aspects, the 5' intron sequence and the 3' intron sequence are derived from at least two different introns. In some aspects, the 5' intron sequence can be a chimeric 5' intron sequence, i.e., a 5' intron sequence containing portions derived from one, two, or more introns. In some aspects, the 3' intron sequence can be a chimeric 3' intron sequence, i.e., a 3' intron sequence containing portions derived from one, two, or more introns. For example, the intron sequence may contain a P1 domain from a first intron, a catalytic core P7 domain from a second intron, and a 3' end from a third intron.

[0189] In some respects, the 5' intron sequence and the 3' intron sequence together correspond to the full-length intron. In some respects, the 5' intron sequence and the 3' intron sequence together are smaller than the full-length intron. In some respects, the 5' intron sequence contains less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, and less than 1%.

[0190] In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of SEQ ID NO: 11-16, 17-22, or 177-178. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of SEQ ID NO: 13 or 19. In some aspects, the 3' intron sequence consists of a nucleotide sequence of any of SEQ ID NO: 11-16, 17-22, or 177-178. In some aspects, the 3' intron sequence consists of a nucleotide sequence of SEQ ID NO: 13 or 19.

[0191] In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of SEQ ID NO: 23-26, 27-30, or 175-176; or a nucleotide sequence of AAA. In some aspects, the 5' intron sequence comprises a nucleotide sequence of AAA. In some aspects, the 5' intron sequence consists of a nucleotide sequence of any of SEQ ID NO: 23-26, 27-30, or 175-176. In some aspects, the 5' intron sequence consists of a nucleotide sequence of AAA.

[0192] In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 11 or 17. In some aspects, the 3' intron sequence consists of the nucleotide sequence of SEQ ID NO: 11 or 17. In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 23 or 27. In some aspects, the 5' intron sequence consists of the nucleotide sequence of SEQ ID NO: 23 or 27. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 11 or 17, and the 5' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 23 or 27. In some aspects, the 3' intron sequence consists of the nucleotide sequence of SEQ ID NO: 11 or 17, and the 5' intron sequence consists of the nucleotide sequence of SEQ ID NO: 23 or 27.

[0193] In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 16 or 22. In some aspects, the 3' intron consists of the nucleotide sequence of SEQ ID NO: 16 or 22. In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 23 or 27. In some aspects, the 5' intron sequence consists of the nucleotide sequence of SEQ ID NO: 23 or 27. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 16 or 22, and the 5' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 23 or 27. In some aspects, the 3' intron sequence consists of the nucleotide sequence of SEQ ID NO: 16 or 22, and the 5' intron sequence consists of the nucleotide sequence of SEQ ID NO: 23 or 27.

[0194] In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 12 or 18. In some aspects, the 3' intron sequence consists of the nucleotide sequence of SEQ ID NO: 12 or 18. In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 24 or 28. In some aspects, the 5' intron sequence consists of the nucleotide sequence of SEQ ID NO: 24 or 28. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 12 or 18, and the 5' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 24 or 28. In some aspects, the 3' intron sequence consists of the nucleotide sequence of SEQ ID NO: 12 or 18, and the 5' intron sequence consists of the nucleotide sequence of SEQ ID NO: 24 or 28.

[0195] In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 13 or 19. In some aspects, the 3' intron sequence consists of the nucleotide sequence of SEQ ID NO: 13 or 19. In some aspects, the 5' intron sequence comprises the nucleotide sequence of AAA. In some aspects, the 5' intron sequence consists of the nucleotide sequence of AAA. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 13 or 19, and the 5' intron sequence comprises the nucleotide sequence of AAA. In some respects, the 3' intron sequence consists of the nucleotide sequence of SEQ ID NO: 13 or 19, and the 5' intron sequence consists of the nucleotide sequence of AAA.

[0196] In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 14 or 20. In some aspects, the 3' intron sequence consists of the nucleotide sequence of SEQ ID NO: 14 or 20. In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 25 or 29. In some aspects, the 5' intron sequence consists of the nucleotide sequence of SEQ ID NO: 25 or 29. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 14 or 20, and the 5' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 25 or 29. In some aspects, the 3' intron sequence consists of the nucleotide sequence of SEQ ID NO: 14 or 20, and the 5' intron sequence consists of the nucleotide sequence of SEQ ID NO: 25 or 29.

[0197] In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 15 or 21. In some aspects, the 3' intron sequence consists of the nucleotide sequence of SEQ ID NO: 15 or 21. In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 26 or 30. In some aspects, the 5' intron sequence consists of the nucleotide sequence of SEQ ID NO: 26 or 30. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 15 or 21, and the 5' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 26 or 30. In some aspects, the 3' intron sequence consists of the nucleotide sequence of SEQ ID NO: 15 or 21, and the 5' intron sequence consists of the nucleotide sequence of SEQ ID NO: 26 or 30.

[0198] In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 177 or 178. In some aspects, the 3' intron sequence consists of the nucleotide sequence of SEQ ID NO: 177 or 178. In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 175 or 176. In some aspects, the 5' intron sequence consists of the nucleotide sequence of SEQ ID NO: 175 or 176. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 177 or 178, and the 5' intron sequence comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 175 or 176. In some aspects, the 3' intron sequence consists of the nucleotide sequence of SEQ ID NO: 177 or 178, and the 5' intron sequence consists of the nucleotide sequence of SEQ ID NO: 175 or 176.

[0199] Non-restrictive examples of 5' intron sequences and 3' intron sequences that can be used in this genetic construct are shown in Table 4.

[0200] Table 4. Exemplary 5' intron sequences and 3' intron sequences.

[0201] In some respects, the 5' intron sequence contains an internal guide sequence. In some respects, the 5' intron sequence consists of an internal guide sequence that is sidelined at 5' with any number of additional intronic nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50, etc.), and / or sidelined at 3' with any number of additional intronic nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50, etc.).

[0202] In some aspects, the 5' intron sequence consists of an internal guide sequence flanking any number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50, etc.) of additional intronic nucleotides at 5', and / or flanking any number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50, etc.) of additional intronic nucleotides at 3'. In some aspects, the 5' intron sequence consists of an internal guide sequence.

[0203] As used herein, the term "intronic nucleotide" refers to a nucleotide that is part of an intron in a derived intron sequence.

[0204] In some respects, the 3' intron sequence contains less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, and less than 1%.

[0205] In some aspects, the 3' intron sequence contains a catalytic core sequence. In some aspects, the 3' intron sequence consists of a catalytic core sequence flanked at 5' by any number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50, etc.) of additional intronic nucleotides, and / or flanked at 3' by any number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50, etc.) of additional intronic nucleotides. In some aspects, the 3' intron sequence consists of a catalytic core sequence flanked at 5' by any number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50, etc.) of additional intronic nucleotides, and / or flanked at 3' by any number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50, etc.). In some aspects, the 3' intron sequence consists of a catalytic core sequence.

[0206] II.B.3. Catalytic Core In some respects, the intron sequence contains the catalytic core. As used herein, the term "catalytic core" refers to a nucleotide sequence containing the sequence and structure essential for catalytic function. The catalytic core interacts with the substrate used in the self-splicing reaction step. The substrate used in the self-splicing reaction step includes exogenous guanosine, 5'-splicing sites, 3'-splicing sites, or combinations thereof.

[0207] In some aspects, the catalytic core comprises sequences of GAG and CC, wherein GAG and CC are separated by (N)n, where N is any one of A, C, G, T, or U, and n is an integer. In some aspects, the catalytic core comprises sequences of ACG and CT (or U), wherein ACG and CT (or U) are separated by (N)n, where N is any one of A, C, G, T, or U, and n is an integer. In some aspects, exogenous G binds to the catalytic core.

[0208] In some respects, the intron sequence does not contain a catalytic core.

[0209] In some respects, the catalytic core contains AGAGA[N]. 6-41 A[N]43-45 GUCC, ACAGA[N] 6-41 A[N] 43-45 GUCG or ACGACU[N] 7-68 The nucleotide sequence of any one of UAGUCU, where N is any one of A, U, C or G, and where [N] 6-41 [N] 43-45 Or [N] 7-68 Each N in each chain can independently be any of A, U, C, or G. In some respects, the catalytic core is composed of AGAGA[N]. 6-41 A[N] 43-45 GUCC, ACAGA[N] 6-41 A[N] 43-45 GUCG or ACGACU[N] 7- 68 The nucleotide sequence of UAGUCU is composed of N, where N is any one of A, U, C, or G, and [N] is […]. 6-41 [N] 43-45 Or [N] 7-68 Each N in each chain can be independently any of A, U, C or G.

[0210] Non-limiting examples of catalytic cores that can be used in this genetic construct are shown in Table 5.

[0211] Table 5. Exemplary Catalytic Cores

[0212] II.B.4. Internal Instruction Sequence (IGS) In some respects, intronic sequences contain internal guide sequences. As used herein, the term "internal guide sequence" or "IGS" refers to a nucleic acid sequence contained within an intronic sequence that pairs with its 5' exon during splicing. In some respects, the internal guide sequence pairs with nucleotides contained within an exon. In some respects, the internal guide sequence is completely or substantially complementary to the nucleotide sequence contained within an exon.

[0213] In some aspects, the internal guide sequence comprises the nucleotide sequence of any one of CTTAA (or CUUAA), CCCTCT (or CCCUCU), agT (or agU), CTT (or CUU), GGT (or GGU), TTGAG (or UUGAG), GGAGGG, tttGct (or uuuGcu), CTGAG (or CUGAG), or GGCC. In some aspects, the internal guide sequence comprises the nucleotide sequence of agT (or agU) or tttGct (or uuuGcu). In some aspects, the internal guide sequence consists of the nucleotide sequence of CTTAA (or CUUAA), CCCTCT (or CCCUCU), agT (or agU), CTT (or CUU), GGT (or GGU), TTGAG (or UUGAG), GGAGGG, tttGct (or uuuGcu), CTGAG (or CUGAG), or GGCC. In some respects, the internal guide sequence consists of a nucleotide sequence of agT (or agU) or tttGct (or uuuGcu).

[0214] Non-restrictive examples of internal guide sequences that can be used in this genetic construct are shown in Table 6.

[0215] Table 6. Exemplary Internal Guidance Sequence

[0216] II.B.5. Homologous Arm In some respects, genetic constructs capable of forming the circular RNAs disclosed herein (i.e., precursor circRNAs or DNA genetic constructs capable of transcribing precursor circRNAs) also include one or more homologous arms. As used herein, the term "homologous arm" refers to a sequence capable of pairing (i.e., hybridizing) with another sequence.

[0217] In some aspects, homologous arms are located at the 5' and / or 3' ends of the genetic constructs disclosed herein. For example, a first homologous arm may be located at the 5' end of the genetic construct, and a second homologous arm may be located at the 3' end of the genetic construct. In some aspects, homologous arm insertions are contained between any two other sequences in the genetic constructs disclosed herein. For example, a first homologous arm may be between a 3' splice site and an exon sequence, and a second homologous arm may be between a 5' splice site and an exon sequence. In some aspects, homologous arms are contained within any other sequences included in the genetic constructs disclosed herein. For example, homologous arms may be contained within intron sequences, exon sequences, splice sites, or any combination thereof. In some aspects, homologous arms may be contained within spacer sequences. As used herein, spacer sequences are any sequences designed to be located between 5' / 3' splice sites and / or functional regions of exon sequences (e.g., IRES, payloads, etc.). In some aspects, homologous arms are excised during circularization and are not contained in the circRNA. In some respects, the homologous arm is not excised during circularization and is included in the circRNA.

[0218] The first and second homologous arms are reverse complementary sequences of the other and are capable of pairing with each other. In some respects, the pairing of the first and second homologous arms alters the spatial conformation of the genetic construct. For example, the pairing of the first and second homologous arms can bring the 3' splice site and the 5' splice site closer together.

[0219] In some aspects, the homology arm comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of any one of SEQ ID NO: 72-73, 76-77, or 171-174. In some aspects, the homology arm consists of a nucleotide sequence of any one of SEQ ID NO: 72-73, 76-77, or 171-174.

[0220] Non-restrictive examples of homologous arms that can be used in this genetic construct are shown in Table 7.

[0221] Table 7. Exemplary Homologous Arms

[0222] In some respects, the 5' intron sequence, 3' intron sequence, 5' splice site, 3' splice site, exon sequence, and optionally homologous arm are located on the same polynucleotide molecule (e.g., on the same DNA or RNA molecule). Therefore, the 5' intron sequence, 3' intron sequence, 5' splice site, 3' splice site, exon sequence, and optionally homologous arm may be in cis configuration (i.e., contained within the same polynucleotide molecule).

[0223] In some respects, one or more exon sequences are sandwiched between 5' and 3' intron sequences. In some respects, the 5' intron sequence is upstream of one or more exon sequences. In some respects, the 3' intron sequence is downstream of one or more exon sequences.

[0224] II.B.6 exon In some respects, the exon sequence (i.e., one or more exons or fragments thereof) contained in an RNA or DNA genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA) contains one exon, or two or more exons.

[0225] In some respects, the exon sequence is (i.e., in the precursor circRNA construct) or encodes (i.e., in the DNA genetic construct capable of transcribing the precursor circRNA) a functional RNA. In some respects, the functional RNA is not a protein-coding RNA. In some respects, the functional RNA is a therapeutic RNA. In some respects, the functional RNA is a protein-coding RNA. In some respects, the protein-coding RNA is translated into a protein. In some respects, the protein is a therapeutic protein.

[0226] In some aspects, RNA or DNA genetic constructs capable of forming the circular RNA disclosed herein (i.e., precursor circRNA or DNA genetic constructs capable of transcribing precursor circRNA) encode one protein. In some aspects, RNA or DNA genetic constructs capable of forming the circular RNA disclosed herein (i.e., precursor circRNA or DNA genetic constructs capable of transcribing precursor circRNA) encode two or more proteins. In some aspects, the sequences encoding two or more proteins may be separated by ribosomal jumping elements or nucleotide sequences encoding protease cleavage sites. Non-limiting examples of self-cleaving 2A peptides are: Thosea-asigna virus 2A peptide (T2A), porcine cyclosporine virus-1 2A peptide (P2A), foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), silkworm (B. mori) cytoplasmic polyhedrosis virus 2A peptide (BmCPV 2A), or silkworm fibrosis virus 2A peptide (BmIFV 2A).

[0227] Non-limiting examples of proteins encoded by RNA or DNA genetic constructs capable of forming circular RNAs disclosed herein (i.e., precursor circRNAs or DNA genetic constructs capable of transcribing precursor circRNAs) are chimeric antigen receptors (CARs), T-cell receptors (TCRs), B-cell receptors (BCRs), immune cell activating or inhibitory receptors, recombinant fusion proteins, chimeric mutant proteins or fusion proteins, or combinations thereof. In some embodiments, therapeutic proteins include antibodies, nanobodies, non-antibody proteins, immunomodulatory ligands, receptors, structural proteins, growth factor ligands or receptors, hormones or hormone receptors, transcription factors, checkpoint inhibitors or agonists, Fc fusion proteins, anticoagulants, coagulation factors, molecular chaperone proteins, antimicrobial proteins, structural proteins, biochemical enzymes, tight junction proteins, mitochondrial stress responses, cytoskeletal proteins, metal-binding proteins, or small molecules. In some embodiments, immunomodulatory ligands include interferons, cytokines, chemokines, or interleukins. In some implementations, the structural protein is a channel protein or nucleoporin, a cytokine, an immune checkpoint inhibitor, an agonist, an antagonist, a chimeric antigen receptor, one or more TCR chains, a secretory T cell or immune cell adaptor, a transcription factor, or an immunosuppressive enzyme.

[0228] In some respects, proteins are cytokines. Non-limiting examples of cytokines are, but are not limited to, IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-35, IL-12p70, IL-27β, IFN-α, IFN-β, IFNγ, TGFβ, or functional fragments thereof.

[0229] In some respects, a protein is an antigen-binding protein or a functional fragment thereof. In some respects, an antigen-binding protein is a trispecific antigen-binding protein (TRITE) or a bispecific antigen-binding protein (BITE) or a functional fragment thereof. In some respects, an antigen-binding protein is an antibody or a functional fragment thereof. Non-limiting examples of antigens include, but are not limited to, Ebola virus glycoprotein, Ebola virus glycoprotein, a4 integrin, β-amyloid protein, β-amyloid fibrils, β-amyloid protein, N3pG (N-terminal truncated form), angiopoietin-like protein 3, Bacillus anthrax PA, B7-H3, B cell maturation antigen, BLyS, C1s, C5, CCR4, CD11a, CD19, CD20, CD22, CD3, CD30, CD33, CD38, CD4, CD52, CD62 (also known as P-selectin), CD79b, CGRP, CGRP receptor, Clostridium difficile. Difficile (Enterotoxin B, Complement 5, CTLA-4, Dabigatran, Ebola virus, EGFR, cMET, Endotoxin, EpCAM, EPCAM / CD3, Factor Ixa, X, FcRn, FGF23, Folate receptor α, G protein-coupled receptor 5D, CD3, GD2, gp100, CD3, GPIIb / IIIa, HER2, IFNAR1, IFNg, IgE, IGF-1R, IL-12 / 23, IL-13, IL-17a, IL-17A, F, IL-17R, IL-1β, IL-23 p19, IL-23p19, IL-2R, IL-36 receptor, IL-4R α, IL-5, IL-5R α, IL-6, IL-6R, LAG-3, MASP-2, cohesin-4, PCSK9, PD-1, PDGFRα, PD-L1, plasma kallikrein, RANK-L, RSV, SARS-CoV-2, sclerosing protein, SLAMF7, thymic stromal lymphopoietin, tissue factor, tissue factor pathway inhibitor, TNF, TROP-2, VEGF, VEGF-A, Ang-2, VEGFR2, von Willebrand factor, and α4β7 integrin. Non-limiting examples of antibodies include, but are not limited to, moromuzumab-CD3, efalizumab, tosimozumab-I131, nebacubitumab, ezolizumab, caputoxumab, daklizumab, olazazumab, acyclovir, rituximab, baliximab, palizumab, infliximab, trastuzumab, adalimumab, teimozumab, omalizumab, cetuximab, bevacizumab, natezumab, panitumumab, ranibizumab, eculizumab, pecelizumab, ustekinumab, canatumab, golimumab, and ofamumab.Tocilizumab, Denosumab, Belimumab, Iprimma, Vibrentuximab, Pertuzumab, Trastuzumab emtansine, Raxicurumab, Oxontocilizumab, Sestoxicumab, Ramucirumab, Vertuzumab, Nivolumab, Pembrolizumab, Belintoolumab, Alenzabumab, Ivolucrata, Idacilizumab, Nexitruzumab, Denutoxicum Anti-, secukinumab, mepolizumab, alexidumab, daratumumab, erlotuzumab, isibetab, rayzumab, belotuzumab, atezolizumab, obbituzumab, brodatumab, duprezumab, oxalitumumab, gusejiruumab, sarriroumab, averuumab, emecizumab, occrezumab, etc. Lalizumab, Duvalimumab, Gefitinib, Ozolimicin, Errenumab, Errenumab-AOE, Ganezumab, Ganezumab-GNLM, Brozosumab, Brozosumab-TWZA, Lanarumab, Lanarumab-Flyo, Moglizumab, Moglizumab-KPKC, Tigrisizumab, Tigrisizumab-ASMN, Remannaizumab, Remannaizumab-VFRM, Revulizumab, Revulizumab-CWVZ, Cimiprizumab, Cimiprizumab-RWLC, Ibalizumab, Ibalizumab-UIYK, Epatamide, Epatamide-LZSG, Parcetumab, Parcetumab-TDFK, Karacizumab, Karacizumab Sildenafil-yhdp, Resalitum, Resalitum-rzaa, Verporutuzumab, Verporutuzumab-piiq, Romozomab, Romozomab-aqqg, Busizumab, Busizumab-dbll, Crizotinib, Crizotinib-tmca, Verentutuzumab, Verentutuzumab-ejfv, Detrituzumab, Detrituzumab-nxki, Ixatuzumab, Ixatuzumab-irfc, Mabelantuzumab, Mabelantuzumab-blmf, Goxatuzumab, Goxatuzumab-hziy, Tancitabine, Tancitabine-cxix, Satralizumab, Satralizumab-mwge, Epranezumab, Epranezumab-jjmr, Ixatuzumab, Epranezumab-jjmr, Epranezumab-dbll, Epranezumab-dbll, Epranezumab-dbll, Epranezumab-tmca, Epranezumab-ejfv, Epranezumab, Epranezumab-nxki, Exatuzumab, Exatuzumab-irfc, Mabelantuzumab, Mabelantuzumab-blmf, Goxatuzumab, Goxatuzumab-hziy, Tancitabine, Tancitabine-cxix, Satralizumab, Satralizumab-mwge, Epranezumab, Epranezumab-jjmr, Epranezumab-dbll, Epranezumab-dbll, Epranezumab-mwge, Epranezumab-jjmr, Epranezumab-dbll, Epranezumab-dbll, Epranezumab-mwge Nelizumab, Inellizumab-cdon, Tiltumab, Tiltumab-trbw, Ivesuzumab, Dotalimab, Dotalimab-gxly, Evantuzumab, Evantuzumab-vmjw, Traroluzumab, Traroluzumab-ldrm, Anilumab, Anilumab-fnia, Tironutuximab, Tironutuximab-lpyl, Atevir, Mateivir, Osimivir-ebgn, Nacitraziumab-gqgk, Macitraziumab-cmkb, Ansumavir-zykl, Adulcanumab, Adulcanumab-avwa, Remdesivir, Sotopixumab, Tissomumab, Tissomumab-tftv, Bimegezumab, Caesarea+IdemetonabTazerulimab, Tazerulimab-ekko, Faraxicillin, Faraxicillin-svoa, Sutemolimab, Sutemolimab-jome, Tesaxagvirimilab, Ciagavirimilab, Pesolilimab, Nicevirimilab, Teliximumab, Teliximumab-mzwv, Utoxilimab, Tebronfos, Tebronfos-tebn, Tadalafil, Mortuzumab, Teritolumab, Trimelimumab, Somituximab Somituximab-gynx, lencanetimab, toripalimab, doxorutuzumab, icoretuzumab, migilizumab, glimetuzumab, parzelizumab, lepritzizumab, taquitozumab, loliximab, cochilizumab, constipation, enometuzumab, sugaryzumab, penprinzim, donepemab, sintilimab, tislelizumab, rivetrazol, nasoribetrazol, and octotozumab.

[0230] In some respects, proteins are chimeric antigen receptors (CARs or CAR-T). Non-limiting examples of CARs include, but are not limited to, CARs containing an antigen-binding domain specific to antigens selected from the group consisting of: CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B cell maturation antigen (BCMA), Tn antigen ((TnAg) or (GalNAca-Ser / Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), and KIT. (CD117), interleukin-13 receptor subunit α-2, mesothelin, interleukin-11 receptor α (IL-11Ra), prostate stem cell antigen (PSCA), serine protease 21, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor β (PDGFR-β), embryonic stage-specific antigen-4 (SSEA-4), CD20, folate receptor α, HER2, HER3, mucin 1, cell surface-associated antigen (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostate enzymes, prostate acid phosphatase (PAP), elongation factor 2 mutant (ELF2M), liver glycoside B2, fibroblast activating protein α (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (precursor, macroprotein factor) β9 subunit (LMP2), glycoprotein 100 (gplOO), oncogene fusion protein (bcr-abl) composed of breakpoint cluster region (BCR) and Abelson mouse leukemia virus oncogene homolog 1 (Abl), tyrosinase, liver glycoside A receptor 2 (EphA2), fucose GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor β, tumor endothelial marker 1(TEM1 / CD248), Tumor endothelial marker 7-related molecules (TEM7R), Tight junction protein 6 (CLDN6), Thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C5 member D (GPRC5D), X chromosome open reading frame 61 (CXORF61), CD97, CD179a, Anaplastic lymphoma kinase (ALK), Polysialic acid, Placenta-specific 1 (PLAC1), GloboH glycoceramide hexasaccharide moiety (GloboH), Breast differentiation antigen (NY-BR-1), Plaque protein 2 (UPK2), Hepatitis A virus cell receptor 1 (HAVCR1), Adrenaline receptor β3 (ADRB3), Pantothecin 3 (PANX3), G protein-coupled receptor 20 (GPR20), Lymphocyte antigen 6 complex locus K9 (LY6K), Olfactory receptor 51E2 (OR51E2), TCRγ alternating reading frame protein (TARP), Wilms tumor protein The following proteins are listed: protein (WT1), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), MAGE family members (including MAGE-A1, MAGE-A3, and MAGE-A4), ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-associated antigen 1, tumor protein p53 (p53), p53 mutant, prostaglandin, serotonin, telomerase, prostate cancer tumor antigen-1, T cell-recognized melanoma antigen 1, rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma apoptosis inhibitor (ML-IAP), and ERG (transmembrane serine protease 2). (TMPRSS2) ETS fusion gene), N-acetylglucosamine transferase V (NA17), pairing box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-associated protein 2 (TRP-2), cytochrome P450 1B1 (CYPIBI), CCCTC binding factor (zinc finger protein)-like protein, T cell recognized squamous cell carcinoma antigen 3 (SART3), pairing box protein Pax-5 (PAX5), acrosome protein-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchoring protein 4(AKAP-4), synovial sarcoma, X-breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renin ubiquitin 1 (RU1), renin ubiquitin 2 (RU2), pod protein, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, heat shock protein 70-2 mutant (mut hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), IgA Fc fragment receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecular-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), mucin-like hormone receptor-like 2 containing EGF-like modules (EMR2), lymphocyte antigen 75 (LY75), phosphatidylinositol proteoglycan-3 (GPC3), Fc receptor-like 5 (FCRL5), MUC16, 5T4, 8H9, anbq integrin, anb Integrin, Alpha-fetoprotein (AFP), B7-H6, Ca-125, CA9, CD44, CD44v7 / 8, CD52, E-cadherin, EMA (epithelial membrane antigen), Epithelial glycoprotein-2 (EGP-2), Epithelial glycoprotein-40 (EGP-40), ErbB4, Epithelial tumor antigen (ETA), Folate-binding protein (FBP), Kinase insertion domain receptor (KDR), κ-light chain, L1 cell adhesion molecule, MFJC18, NKG2D, Carcinoma-fetal antigen (h5T4), Tumor / testis antigen IB, GAGE, GAGE-1, BAGE, SCP-1, CTZ9, SAGE, CAGE, CT10, MART-1, Immunoglobulin λ-like polypeptide 1 (IGLL1), Hepatitis B surface antigen-binding protein (HBsAg), Viral capsid antigen (VCA), Early antigen (EA), EBV nuclear antigen (EBNA), HHV-6 p41 early antigen, HHV-6B U94 latent antigen, HHV-6B p98 late antigen, cytomegalovirus (CMV) antigen, large T antigen, small T antigen, adenovirus antigen, respiratory syncytial virus (RSV) antigen, hemagglutinin (HA), neuraminidase (NA), parainfluenza type 1 antigen, parainfluenza type 2 antigen, parainfluenza type 3 antigen, parainfluenza type 4 antigen, human metapneumovirus (HMPV) antigen, hepatitis C virus (HCV) core antigen, HIV p24 antigen, human T-cell lymphotrophic virus (HTLV-1) antigen, Merkel cell polyomavirus small T antigen, Merkel cell polyomavirus large T antigen, Kaposi's sarcoma-associated herpesvirus (KSHV) lysate nuclear antigen, and KSHV latent nuclear antigen.

[0231] In some respects, proteins are transcription factors. Non-limiting examples of transcription factors are, but are not limited to, forkhead box protein P3 (Foxp3) and signal transduction and transcriptional activation proteins (STATs, such as STAT1, STAT2, STAT3, STAT4, STAT5 (including STAT5A and STAT5B) and STATE).

[0232] II.B.7. IRES In some respects, the RNA or DNA constructs disclosed herein also contain internal ribosome entry sites (IRES). In some respects, the IRES are contained within exons.

[0233] The IRES, initially identified as a characteristic small RNA viral RNA, plays a crucial role in initiating protein synthesis in the absence of a 5' cap structure. The IRES can act as a single ribosome binding site or as one of multiple ribosome binding sites for mRNA. Nucleic acids or mRNAs containing more than one functional ribosome binding site can encode several peptides or polypeptides that are independently translated by ribosomes (“polycistronic nucleic acid molecules”). When an IRES is provided to a nucleic acid or mRNA, a second translatable region is optionally further provided.

[0234] Examples of IRES sequences that may be used according to this disclosure include, but are not limited to, those from piconeseviruses (e.g., FMDV), insect pest virus (CFFV), poliovirus (PV), encephalomyelitis virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukosis virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV). In some aspects, the genetic constructs disclosed herein comprise IRES. In some embodiments, the genetic constructs disclosed herein comprise IRES having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequence of SEQ ID NO: 87-110 or 145-168. This document discloses modifications to IRES and associated sequences to increase or decrease IRES activity, for example, by truncating the 5' and / or 3' ends of the IRES, adding a 5' spacer region to the IRES, modifying the 5' translation initiation site by 6 nucleotides (Kozak sequence), modifying alternative translation initiation sites, and creating chimeric / hybrid IRES sequences. In some aspects, the IRES sequences in the genetic constructs disclosed herein include one or more of these modifications relative to the natural IRES (e.g., the natural IRES disclosed in Table 8 (SEQ ID NO: 87-110 or 145-168)).

[0235] Table 8. Exemplary IRES

[0236] In some respects, the inclusion of IRES allows translation of one or more open reading frames (e.g., open reading frames contained in exon sequences) from circular RNA. IRES elements attract the eukaryotic ribosomal translation initiation complex and promote translation initiation. See, for example, Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20: 102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques 1997 22 150-161. Many IRES sequences are available and include sequences derived from a variety of viruses, such as leader sequences from piconemaviruses, such as encephalomyocarditis virus (EMCV) UTR (Jang et al., J. Virol. (1989) 63: 1651-1660), polio virus leader sequences, hepatitis A virus leader sequences, hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25): 15125-15130), IRES elements from foot-and-mouth disease virus (Ramesh et al., Nucl. AcidRes. (1996) 24:2697-2700), Giardia virus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), etc.

[0237] In some respects, IRES is the following IRES sequence: Taura syndrome virus, triatomine virus, Tyler encephalomyelitis virus, simian virus 40, red imported fire ant virus 1 (Solenopsis invicta virus 1), Rhopalosiphum padi virus, reticuloendotheliosis virus, human poliovirus 1, stink bug enterovirus, Kashmir bee virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Himetobi P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, equine rhinitis virus, tea geometrid moth picorna-like virus, encephalocarditis virus, fruit fly C virus (Drosophila C) Virus), human Coxsackievirus B3, Tobacco Mosaic Virus (Cruciflora), Cricket Paralysis Virus, Bovine Viral Diarrhea Virus 1, Black Bee Queen Cell Virus, Aphid Lethal Paralysis Virus, Avian Encephalomyelitis Virus, Acute Bee Paralysis Virus, Hibiscus Chlorotic Ringspot Virus, Classical Swine Fever Virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila Antennae and Leg Gene (Drosophila) (antennapedia), human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIFlα, human n.myc, mouse Gtx, human p27kip1, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila harvester gene, canine Scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless gene, Saccharomyces cerevisiae TFIID(S).Cerevisiae TFIID), Saccharomyces cerevisiae YAP1, Tobacco etch virus, Turnip wrinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Small double RNA virus, HCV QC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivary virus A SHI, Salivary virus FHB, Salivary virus NG-J1, Human paraenteritis virus 1, Crohn's virus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A A) Pasi virus A2, Echo virus E14, Human paraenterovirus 5, Aichi virus, Hepatitis A virus HA16, Phocotic hepatitis virus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-CK1737, GBV-C Iowa, Pegivirus A 1220, Pasi virus A 3, Sapello virus, Rosa virus B, Bakunsa virus, Tremor virus A, Porcine Pasi virus 1, PLV-CHN, Pasi virus A, Sicinivirus, Hepatitis virus K, Hepatitis virus A, BVDV1, Border disease virus, BVDV2, CSFV-PK15C, SF573 bicistronic virus, Hubei small ribonucleic acid-like virus, CRPV, Salivary virus A BN5, Salivary virus A Aptamers of BN2, salivary virus A02394, salivary virus A GUT, salivary virus A CH, salivary virus A SZ1, salivary virus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G.

[0238] II.B.8. UTR and Kozak sequences In some respects, the genetic constructs disclosed herein also contain a UTR. For example, in some respects, the precursor circRNA or circRNA generated as disclosed herein contains a payload sequence, wherein the payload sequence contains a UTR. In some respects, the UTR is a 5'-UTR. In some respects, the UTR is a 3'-UTR. In some respects, the UTR contains both a 5'-UTR and a 3'-UTR.

[0239] The untranslated region (UTR) of a gene is transcribed but not translated. The 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon; while the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal. Increasing evidence suggests a regulatory role of the UTR in the stability of nucleic acid molecules and in translation. Therefore, when the precursor circRNA or circRNA produced as disclosed herein contains a UTR, the stability of the payload sequence is increased, for example, compared to sequences without a UTR. As described herein, in some respects, increased stability leads to increased expression of the encoded protein. Native 5'-UTRs have features that play a role in translation initiation. They contain features like the Kozak sequence, which is well-known for its involvement in ribosome-initiated translation of many genes. The Kozak sequence has a consistent CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". It is also known that 5'-UTRs form secondary structures involved in elongation factor binding.

[0240] The 5'-UTR secondary structures involved in elongation factor binding can interact with other RNA-binding molecules in the 5'-UTR or 3'-UTR to regulate gene expression. For example, the binding of the elongation factor EIF4A2 to secondary structural elements in the 5'-UTR is essential for microRNA-mediated repression (Meijer HA et al., Science, 2013, 340, 82-85, which is incorporated herein by reference in its entirety). Different secondary structures in the 5'-UTR can be incorporated into flanking regions to stabilize or selectively destabilize mRNA in specific tissues or cells.

[0241] By engineering features found in genes that are typically abundantly expressed in specific target organs, humans can enhance the stability of nucleic acid sequences (e.g., the payload sequences of the synthetic circuits provided herein) and protein production. For example, introducing the 5'-UTR of liver-expressed mRNAs (such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII) can be used to enhance the expression of nucleic acid molecules (such as mRNA) in hepatocyte lines or the liver. Similarly, it is possible to improve expression in a tissue by using the 5'-UTR of other tissue-specific mRNAs—for muscle (MyoD, myosin, myoglobin, myopoietin, Herculin), for endothelial cells (Tie-1, CD36), for myeloid cells (C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin), and for lung epithelial cells (SP-A / B / C / D).

[0242] Other non-UTR sequences can also be incorporated into the UTR (e.g., 5'-UTR and / or 3'-UTR). For example, introns or portions of intron sequences can be incorporated into the flanking regions of nucleic acid sequences (e.g., the payload sequences of the synthetic circuits provided herein).

[0243] In some respects, one or more nucleotides within the UTR (e.g., the 5'-UTR and / or the 3'-UTR) may be mutated, substituted, and / or removed. For example, one or more nucleotides upstream of the start codon may be substituted with another nucleotide. The one or more nucleotides to be substituted may be located at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 nucleotides upstream of the start codon. As another example, one or more nucleotides upstream of the start codon may be removed from the UTR. In some respects, the genetic constructs disclosed herein contain the Kozak sequence.

[0244] 3'-UTRs are known to contain embedded adenosine and uridine segments. These AU-rich features are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be classified into three classes (Chen et al., 1995): Class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this class of AREs include GM-CSF and TNF-α. Class III AREs are less clearly defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myopoietin are two well-studied examples in this class. Most proteins that bind to AREs are known to destabilize messengers, while members of the ELAV family, most notably HuR, have been shown to increase mRNA stability. HuR binds to all three classes of AREs. Engineering HuR-specific binding sites into the 3'-UTR of nucleic acid molecules can lead to HuR binding, thereby stabilizing in vivo information.

[0245] In some respects, the introduction, removal, or modification of AU-rich elements (AREs) in the 3'-UTR can be used to regulate the stability of nucleic acid sequences. When engineering specific genetic constructs disclosed herein, one or more copies of AREs can be introduced to make the nucleic acid sequence less stable and thus restrict translation and reduce the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability and thereby increase the translation and production of the resulting protein.

[0246] In some respects, the genetic constructs disclosed herein contain a 3' UTR. In some embodiments, the 3' UTR is derived from human β-globin, human α-globin, or Xenopus laevis. (Xenopus) β-globin, African clawed toad α-globin, human prolactin, human GAP-43, human eEFlal, human Tau, human TNFα, dengue virus, Hantavirus small mRNA, Bunyavirus small mRNA, turnip mosaic virus, hepatitis C virus, rubella virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH, human microtubules, hibiscus chlorotic ringspot virus, marmot hepatitis virus post-translational regulatory elements, Sindbis virus, turnip shrunken virus, tobacco etched virus, or Venezuelan equine encephalitis virus.

[0247] In some respects, the genetic constructs disclosed herein contain a 5' UTR. In some embodiments, the 5' UTR is derived from human β-globin, Xenopus laevis, or other sources. (Xenopus laevis) β-globin, human α-globin, African clawed toadAlpha globin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etch virus, turnip wrinkle virus, or adenovirus triple leader sequence.

[0248] II.B.9. Interval In some aspects, the genetic constructs disclosed herein include spacer sequences. In some aspects, the genetic constructs disclosed herein include spacer sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleotide sequences of SEQ ID NO: 74-75 or 78-79. In some aspects, the genetic constructs disclosed herein include spacer regions composed of the nucleotide sequences of SEQ ID NO: 74-75 or 78-79.

[0249] Non-restrictive examples of spacer sequences that can be used in this genetic construct are shown in Table 9.

[0250] Table 9. Exemplary Spacing Zones

[0251] In some aspects, the genetic constructs disclosed herein include first (5') and / or second (3') spacer regions. In some aspects, including a spacer region between the 3' intron fragment and the IRES can improve splicing efficiency by preserving secondary structures in these regions to prevent their interaction. In some aspects, the first (between the 3' group fragment and the IRES) and second (between the expressed sequence and the 5' intron fragment) spacer regions contain additional base-pairing regions that are expected to pair bases with each other without pairing with the first and second duplex-forming regions. In some aspects, such spacer region base pairing brings intron fragments closer together, further improving splicing efficiency. In some aspects, the combination of base pairing between the first and second duplex-forming regions and base pairing between the first and second spacer regions individually promotes the formation of splice bubbles containing intron fragments flanked by adjacent base-pairing regions. In some respects, a spacer region is a contiguous sequence having one or more of the following characteristics: 1) predicts avoidance of interference with proximal structures, such as IRES, expressed sequences, or introns; 2) is at least 7 nt long and no more than 100 nt; 3) is located after and adjacent to a 3' intron and / or before and adjacent to a 5' intron; and 4) contains one or more of the following regions: a) an unstructured region at least 5 nt long, b) a region at least 5 nt long that pairs with the distal sequence bases, including another spacer region, and c) a structured region at least 7 nt long, the extent of which is limited to the sequence of the spacer region. A spacer region may have several regions, including unstructured regions, base-pairing regions, hairpin / structured regions, and combinations thereof. In some respects, a spacer region has a structured region with a high GC content. In some respects, a region within a spacer region pairs with a region within the same spacer region. In some respects, a region within a spacer region pairs with a region within another spacer region. In some respects, a spacer region contains one or more hairpin structures. In some aspects, the spacer region comprises one or more hairpin structures having a stem of 4 to 12 nucleotides and a loop of 2 to 10 nucleotides. In some aspects, an additional spacer region exists between the 3' intron fragment and the IRES. In some aspects, this additional spacer region prevents the structured regions of the IRES from interfering with the folding of the 3' intron fragment or reduces the extent to which this occurs. In some aspects, the 5' spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides in length. In some aspects, the 5' spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some aspects, the 5' spacer sequence is 5 to 50, 10 to 50, 20 to 50, 20 to 40, and / or 25 to 35 nucleotides in length.In some respects, the 5' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some respects, the 5' spacer sequence is a polyA sequence. In some respects, the 5' spacer sequence is a polyAC sequence. In some respects, the spacer region contains approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polyAC content. In some respects, the spacer contains approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% polypyrimidine (C / T or C / U) content.

[0252] II.B.10. Poly(A) In some aspects, the genetic constructs disclosed herein also include a polyA region. In some aspects, the polyA region is at least 15, 30, or 60 nucleotides long. In some aspects, the polyA sequence is removed during circularization. Therefore, oligonucleotides hybridizing with the polyA sequence (such as deoxythymidine oligonucleotides (oligo(dT)) conjugated to a solid surface (e.g., resin)) can be used to separate circular RNA from its precursor RNA. Other sequences can also be set at the 5' of a 3' intron fragment or the 3' of a 5' intron fragment, and complementary sequences can be similarly used for circular RNA purification. In some aspects, the polyA region contains a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 85 or 86. In some aspects, the polyA region consists of SEQ ID NO: 85 or 86.

[0253] Non-restricted examples that can be used in the polyA region of this genetic construct are shown in Table 10.

[0254] Table 10. Exemplary polyA regions

[0255] II.B.11. Scar excision In some aspects, the RNA or DNA genetic constructs disclosed herein also include one or more nucleotide sequences derived from an organism derived from an intron or exon sequence. In some aspects, the one or more nucleotide sequences derived from an organism derived from an intron or exon sequence are intron sequences, exon sequences, or any combination thereof. In some aspects, the one or more nucleotide sequences derived from an organism derived from an intron or exon sequence are located near a splice site. In some aspects, the one or more nucleotide sequences derived from an organism derived from an intron or exon sequence are adjacent to a splice site. In some aspects, the one or more nucleotide sequences derived from an organism derived from an intron or exon sequence are not excised from the precursor circRNA during splicing. In these aspects, when referring to those sequences contained in a precursor circRNA or in a DNA construct capable of transcribing a precursor circRNA, the one or more nucleotide sequences derived from an organism derived from an intron or exon sequence are also referred to herein as pre-excision scars, and when referring to those sequences contained in a circRNA, as excision scars.

[0256] In some aspects, the excision scar comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 80-81 or 82-83. In some aspects, the excision scar consists of SEQ ID NO: 80-81 or 82-83.

[0257] Non-limiting examples of excision scars that can be used for this genetic construct are shown in Table 11.

[0258] Table 11. Exemplary Excision Scars

[0259] II.B.12. Promoter In some respects, the RNA or DNA genetic constructs disclosed herein also include a promoter. In some respects, the promoter is removed during circularization. In some respects, the promoter is T7, T3, or SP6. In some respects, the promoter is a T7 promoter. In some respects, the promoter is a T3 promoter. In some respects, the promoter is an SP6 promoter. In some respects, the promoter comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any of SEQ ID NO: 133-138. In some respects, the promoter consists of a nucleotide sequence of any of SEQ ID NO: 133-138.

[0260] Non-restrictive examples of promoters that can be used in this genetic construct are shown in Table 12.

[0261] Table 12. Exemplary promoters

[0262] II.C. Exemplary Constructs Exemplary embodiments of the genetic constructs disclosed herein are described in detail below. For simplicity, exemplary genetic constructs are preferably described with reference to RNA genetic constructs (i.e., precursor circRNAs). It should be understood that DNA genetic constructs capable of transcribing such precursor circRNAs should also be considered to be included in the following description. Thus, for example, any RNA nucleotide sequence listing uracil (U) should be understood to encompass the corresponding DNA nucleotide sequence listing thymine (T).

[0263] II.C.1. Exemplary Genetic Construct A In some respects, a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA) comprises the following formula: 5'-PHA-3S-E-5S-B-H'-3', wherein: P contains a promoter (e.g., T7, T3, SP6, etc.), H contains a 5' homologous arm, A contains a 3' intron sequence, 3S contains a 3' splice site, E contains an exon sequence (i.e., one or more exons or fragments thereof), 5S contains a 5' splice site, B contains a 5' intron sequence, H' contains a 3' homologous arm, and wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct. In some respects, the 5' and 3' intron sequences are reverse complementary to each other.

[0264] In some aspects, the intron sequence comprises a nucleotide sequence derived from a group I intron. In some aspects, the group I intron comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 1-5 or 6-10. In some aspects, the group I intron consists of SEQ ID NO: 1-5 or 6-10.

[0265] In some aspects, the 5' intron sequence contains a nucleotide sequence derived from group I introns. In some aspects, the 5' intron sequence contains an internal guide site (IGS). In some aspects, the 5' intron sequence consists of an internal guide site (IGS). In some aspects, the IGS contains a nucleotide sequence of any of the following: CTTAA (or CUUAA), CCCTCT (or CCCUCU), agT (or agU), CTT (or CUU), GGT (or GGU), TTGAG (or UUGAG), GGAGGG, tttGct (or uuuGcu), CTGAG (or CUGAG), or GGCC. In some aspects, the internal guide sequence contains a nucleotide sequence of agT (or agU) or tttGct (or uuuGcu). In some aspects, the IGS consists of any one of CTTAA (or CUUAA), CCCTCT (or CCCUCU), agT (or agU), CTT (or CUU), GGT (or GGU), TTGAG (or UUGAG), GGAGGG, tttGct (or uuuGcu), CTGAG (or CUGAG), or GGCC. In some aspects, the internal guide sequence consists of any one of agT (or agU) or tttGct (or uuuGcu). In some aspects, the 5' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 23-26 or 27-30, or a nucleotide sequence of AAA. In some aspects, the 5' intron sequence consists of SEQ ID NO: 23-26, or consists of a nucleotide sequence of AAA.

[0266] In some aspects, the 3' intron sequence contains nucleotide sequences derived from group I introns. In some aspects, the 3' intron sequence contains a catalytic core. In some aspects, the catalytic core contains sequences of GAG and CC, wherein GAG and CC are separated by (N)n, where N is any one of A, C, G, T, or U, and n is an integer. In some aspects, the catalytic core contains sequences of ACG and CT (or U), wherein ACG and CT (or U) are separated by (N)n, where N is any one of A, C, G, T, or U, and n is an integer.

[0267] In some respects, the catalytic core contains AGAGA[N]. 6-41 A[N] 43-45 GUCC, ACAGA[N] 6-41 A[N] 43-45 GUCG or ACGACU[N] 7-68The nucleotide sequence of any one of UAGUCU, where N is any one of A, U, C or G, and where [N] 6-41 [N] 43- 45 or [N] 7- Each N in each chain of 68 can independently be any of A, U, C, or G. In some respects, the catalytic core is composed of AGAGA[N]. 6-41 A[N] 43-45 GUCC, ACAGA[N] 6-41 A[N] 43-45 GUCG or ACGACU[N] 7- 68 The system consists of UAGUCU, where N is any one of A, U, C, or G, and [N] is a subset of U. 6-41 [N] 43- 45 or [N] 7- Each N in each strand of 68 can independently be any one of A, U, C, or G. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 11-15 or 17-21. In some aspects, the 3' intron sequence consists of SEQ ID NO: 11-15 or 17-21.

[0268] In some respects, the 5' intron sequence and the 3' intron sequence together are smaller than the complete intron.

[0269] In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 23 or 27. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 11 or 17. In some respects, the 5' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 23 or 27, and the 3' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 11 or 17.

[0270] In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 24 or 28. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 12 or 18. In some respects, the 5' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 24 or 28, and the 3' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 12 or 18.

[0271] In some aspects, the 5' intron sequence contains the nucleotide sequence of AAA. In some aspects, the 3' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 13 or 19. In some aspects, the 5' intron sequence contains the nucleotide sequence of AAA, and the 3' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 13 or 19.

[0272] In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 25 or 29. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 14 or 20. In some respects, the 5' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 25 or 29, and the 3' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 14 or 20.

[0273] In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 26 or 30. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 15 or 21. In some respects, the 5' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 26 or 30, and the 3' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 15 or 21.

[0274] In some aspects, the 3' splice site comprises two nucleotides, one upstream and one downstream. In some aspects, the upstream nucleotide is G and the downstream nucleotide is N, where N is any one of A, C, G, T, or U. In some aspects, N is C. In some aspects, the 3' splice site comprises a sequence of formula 5'-GN-3', wherein the splice site is between G and N, and wherein N is any one of A, C, G, T, or U. In some aspects, the 3' splice site comprises a sequence of formula 5'-GC-3'.

[0275] In some aspects, the 5' splice site comprises two nucleotides, one upstream and one downstream. In some aspects, the upstream nucleotide is U or T, and the downstream nucleotide is N, where N is any one of A, C, G, T, or U. In some aspects, N is A. In some aspects, the 5' splice site comprises a sequence of the formula 5'-U (or T)-N-3', where the splice site is between U (or T) and N, and where N is any one of A, C, G, T, or U. In some aspects, the 5' splice site comprises a sequence of the formula 5'-U (or T)-A-3'.

[0276] In some aspects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 31-35. In some aspects, the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 37-40, or a nucleotide sequence of NAGU ^AAA. In some respects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 31-35, and the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 37-40, or a nucleotide sequence of NAGU ^AAA.

[0277] In some aspects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 31. In some aspects, the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 37. In some respects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 31, and the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 37.

[0278] In some aspects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 32. In some aspects, the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 38. In some respects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 32, and the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 38.

[0279] In some aspects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 33. In some aspects, the 5' splice site comprises a nucleotide sequence of NAGU ^AAA. In some aspects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 33, and the 5' splice site comprises a nucleotide sequence of NAGU ^AAA.

[0280] In some aspects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 34. In some aspects, the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 39. In some respects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 34, and the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 39.

[0281] In some aspects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 35. In some aspects, the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 40. In some respects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 35, and the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 40.

[0282] In some aspects, the 5' homologous arm comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 72 or 76. In some aspects, the 3' homologous arm comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 73 or 77.

[0283] In some respects, the 5' splice site is located at the boundary between the 5' intron sequence and the exon sequence. In some respects, the 3' splice site is located at the boundary between the 3' intron sequence and the exon sequence. In some respects, the intron sequence is completely excised from the RNA genetic construct (i.e., the precursor circRNA). In some respects, the resulting circRNA does not contain any intron sequence. In some respects, the resulting circRNA does not contain one or more intron sequence residues. In some respects, the resulting circRNA does not contain one or more nucleotide sequences derived from organisms derived from the 5' and 3' intron sequences, i.e., the resulting circRNA does not contain "excision scars".

[0284] In some respects, precursor circRNAs can form circular RNAs. In some respects, precursor circRNAs can form circular RNAs in the presence of exogenous G. In some respects, precursor circRNAs can form circular RNAs in which the circular RNA does not contain any sequences derived from organisms derived from the 5' intron sequence and the 3' intron sequence ("excision scar").

[0285] In some respects, the genetic construct from 5' to 3' includes a 5' homologous arm, a 3' intron sequence, a 3' splice site, polyA, IRES, one or more exons, polyA, a 5' intron sequence containing an internal guide sequence (IGS), a 5' splice site, and H' contains a 3' homologous arm, wherein the 3' intron sequence contains a catalytic core, and wherein the RNA construct is capable of forming a circular RNA that does not contain any sequence derived from the genome from which the 5' and 3' intron sequences are derived ("excision scars").

[0286] II.C.2. Exemplary Genetic Construct B In some respects, a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA) comprises the following formula: 5'-PHA-3S-E-5S-B-H'-3', wherein: P contains a promoter (e.g., T7, T3, SP6, etc.), H contains a 5' homologous arm, A contains an intron sequence, 3S contains a 3' splice site, E contains an exon sequence containing a first excisional scar precursor (X) and a second excisional scar precursor X', B contains a 5' intron sequence, and H' contains a 3' homologous arm; and wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct. In some respects, the 5' and 3' intron sequences are reverse complementary to each other.

[0287] In some aspects, the intron sequence comprises a nucleotide sequence derived from a group I intron. In some aspects, the group I intron comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 5 or 10. In some aspects, the group I intron consists of SEQ ID NO: 5 or 10.

[0288] In some aspects, the 5' intron sequence contains an internal guide site (IGS). In some aspects, the IGS contains a nucleotide sequence of GGT (or GGU) or GGCC. In some aspects, the 5' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 26 or 30. In some aspects, the 5' intron sequence consists of SEQ ID NO: 26 or 30.

[0289] In some respects, the 3' intron sequence contains nucleotide sequences derived from group I introns. In some respects, the catalytic core contains ACGACU[N]. 7-68 The nucleotide sequence of UAGUCU, where N is any one of A, U, C or G, and where [N] 6-41 [N] 43- 45 or [N] 7- Each N in each chain of 68 can independently be any of A, U, C, or G. In some respects, the catalytic core is composed of ACGACU[N]. 7-68 The system consists of UAGUCU, where N is any one of A, U, C, or G, and [N] is a subset of U. 6-41 [N] 43-45 or [N] 7- Each N in each strand of 68 can independently be any one of A, U, C, or G. In some respects, the 3' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 15 or 21.

[0290] In some respects, the 5' intron sequence and the 3' intron sequence together are smaller than the complete intron.

[0291] In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 26 or 30. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 15 or 21. In some respects, the 5' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 26 or 30, and the 3' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 15 or 21.

[0292] In some aspects, the 3' splice site comprises two nucleotides, one upstream and one downstream. In some aspects, the upstream nucleotide is G and the downstream nucleotide is N, where N is any one of A, C, G, T, or U. In some aspects, N is C. In some aspects, the 3' splice site comprises a sequence of formula 5'-GN-3', wherein the splice site is between G and N, and wherein N is any one of A, C, G, T, or U. In some aspects, the 3' splice site comprises a sequence of formula 5'-GC-3'.

[0293] In some aspects, the 5' splice site comprises two nucleotides, one upstream and one downstream. In some aspects, the upstream nucleotide is U or T, and the downstream nucleotide is N, where N is any one of A, C, G, T, or U. In some aspects, N is A. In some aspects, the 5' splice site comprises a sequence of the formula 5'-U (or T)-N-3', where the splice site is between U (or T) and N, and where N is any one of A, C, G, T, or U. In some aspects, the 5' splice site comprises a sequence of the formula 5'-U (or T)-A-3'.

[0294] In some aspects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 36. In some aspects, the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 41. In some respects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 36, and the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 41.

[0295] In some aspects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with the 3' intron sequence derived from Synechococcus PCC 6301 tRNA (LEU), as shown in SEQ ID NO: 41. In some aspects, the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with the 5' intron sequence derived from Synechococcus PCC 6301 tRNA (LEU), as shown in SEQ ID NO: 41. In some respects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with the 3' intron sequence derived from Synechococcus PCC 6301 tRNA (LEU) as shown in SEQ ID NO: 41, and the 5' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with the 5' intron sequence derived from Synechococcus PCC 6301 tRNA (LEU) as shown in SEQ ID NO: 41.

[0296] In some aspects, the 5' homologous arm comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 72 or 76. In some aspects, the 3' homologous arm comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 73 or 77.

[0297] In some aspects, the 5' splice site is not at the boundary between the 5' intron sequence and the exon sequence. In some aspects, the 3' splice site is not at the boundary between the 3' intron sequence and the exon sequence. In some aspects, the intron sequence is not completely excised from the RNA genetic construct (i.e., the precursor circRNA). In some aspects, the resulting circRNA contains at least a portion of the intron sequence. In some aspects, the resulting circRNA contains one or more intron sequence residues. In some aspects, the resulting circRNA contains one or more nucleotide sequences derived from organisms derived from the 5' and 3' intron sequences, i.e., the resulting circRNA contains at least one "excision scar". In some aspects, the excision scar contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with any of SEQ ID NO: 80-81 or 82-83. In some respects, the excision scar consists of any one of SEQ ID NO: 80-81 or 82-83.

[0298] In some respects, precursor circRNAs can form circular RNAs. In some respects, precursor circRNAs can form circular RNAs in the presence of exogenous G. In some respects, precursor circRNAs can form circular RNAs containing excision scars.

[0299] II.C.3. Exemplary Genetic Construct C In some respects, a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA) comprises the following formula: 5'-PA-3S-E-3', wherein P contains a promoter (e.g., T7, T3, SP6, etc.), A contains a 3' intron sequence, 3S contains a 3' splice site, and E contains an exon sequence containing a catalytic nucleotide (CN); and wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct.

[0300] In some aspects, the intron sequence comprises a nucleotide sequence derived from a group I intron. In some aspects, the group I intron comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 1, 3-5; or 6, 8-10. In some aspects, the group I intron consists of SEQ ID NO: 1, 3-5; or 6, 8-10.

[0301] In some aspects, the 3' intron sequence contains a catalytic core. In some aspects, the catalytic core contains sequences of GAG and CC, wherein GAG and CC are separated by (N)n, where N is any one of A, C, G, T, or U, and n is an integer. In some aspects, the catalytic core contains sequences of ACG and CT (or U), wherein ACG and CT (or U) are separated by (N)n, where N is any one of A, C, G, T, or U, and n is an integer. In some aspects, the catalytic core contains AGAGA[N]. 6-41 A[N] 43-45 GUCC or ACGACU[N] 7-68 The nucleotide sequence of UAGUCU, where N is any one of A, U, C or G, and where [N] 6-41 [N] 43- 45 or [N] 7- Each N in each chain of 68 can independently be any of A, U, C, or G. In some respects, the catalytic core is composed of AGAGA[N]. 6-41 A[N] 43-45 GUCC or ACGACU[N] 7-68 The system consists of UAGUCU, where N is any one of A, U, C, or G, and [N] is a subset of U. 6-41 [N] 43- 45 or [N] 7- Each N in each strand of 68 can independently be any one of A, U, C, or G. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with any of SEQ ID NO: 13-16 or 19-22. In some aspects, the 3' intron sequence consists of any of SEQ ID NO: 13-16 or 19-22.

[0302] In some respects, the 3' intron sequence is smaller than the complete intron.

[0303] In some aspects, the 3' splice site comprises two nucleotides, one upstream of the splice site and the other downstream of the splice site. In some aspects, the upstream nucleotide is G and the downstream nucleotide is N, wherein N is any one of A, C, G, T, or U. In some aspects, N is C. In some aspects, the 3' splice site comprises a sequence of formula 5'-GN-3', wherein the splice site is between G and N, and wherein N is any one of A, C, G, T, or U. In some aspects, the 3' splice site comprises a sequence of formula 5'-GC-3'. In some aspects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 31 or 33-35. In some respects, the 3' splice site consists of SEQ ID NO: 31 or 33-35.

[0304] In some respects, the 5' homologous arm contains a nucleotide sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 72 or 76.

[0305] In some respects, the 3' splice site is located at the boundary between the 3' intron sequence and the exon sequence. In some respects, the intron sequence is completely excised from the RNA genetic construct (i.e., the precursor circRNA). In some respects, the resulting circRNA does not contain any intron sequence. In some respects, the resulting circRNA does not contain one or more intron sequence residues. In some respects, the resulting circRNA does not contain one or more nucleotide sequences derived from organisms derived from the 3' intron sequence, i.e., the resulting circRNA does not contain an "excision scar".

[0306] In some respects, precursor circRNAs can form circular RNAs. In some respects, precursor circRNAs can form circular RNAs in the presence of exogenous G. In some respects, precursor circRNAs can form circular RNAs where the circular RNA does not contain excision scars.

[0307] II.C.4. Exemplary Genetic Construct D In some respects, a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA) comprises the following formula: 5'-PA-3S-E-3', wherein P contains a promoter (e.g., T7, T3, SP6, etc.), A contains a 3' intron sequence, and E contains an exon sequence (E) comprising a first part (E'), a first homologous arm (HI), a second part (E''), a second homologous arm (HI'), and a third exon sequence (E''') containing a catalytic nucleotide (CN); and wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct.

[0308] In some aspects, the intron sequence comprises a nucleotide sequence derived from a group I intron. In some aspects, the group I intron comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 1 or 6. In some aspects, the group I intron consists of SEQ ID NO: 1 or 6.

[0309] In some respects, the 3' intron sequence contains a catalytic core. In other respects, the catalytic core contains AGAGA[N]. 6-41 A[N] 43-45 A sequence of GUCCs, where N is any one of A, U, C, or G, and where [N] 6-41 Or [N] 43-45 Each N in each chain can independently be any of A, U, C, or G. In some respects, the catalytic core is composed of AGAGA[N]. 6-41 A[N] 43-45 The GUCC structure is composed of N, where N is any one of A, U, C, or G, and [N] is... 6-41 Or [N] 43-45 Each N in each strand can independently be any one of A, U, C, or G. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with any of SEQ ID NO: 11 or 17. In some aspects, the 3' intron sequence consists of any of SEQ ID NO: 11 or 17.

[0310] In some respects, the 3' intron sequence is smaller than the complete intron.

[0311] In some aspects, the 3' splice site comprises two nucleotides, one upstream of the splice site and the other downstream of the splice site. In some aspects, the upstream nucleotide is G and the downstream nucleotide is N, wherein N is any one of A, C, G, T, or U. In some aspects, N is A. In some aspects, the 3' splice site comprises a sequence of formula 5'-GN-3', wherein the splice site is between G and N, and wherein N is any one of A, C, G, T, or U. In some aspects, the 3' splice site comprises a sequence of formula 5'-GA-3'. In some aspects, the 3' splice site comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 169 or 170. In some respects, the 3' splice site consists of SEQ ID NO: 169 or 170.

[0312] In some aspects, the first homologous arm comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 171 or 172. In some aspects, the second homologous arm comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 173 or 174.

[0313] In some respects, the 3' splice site is located at the boundary between the 3' intron sequence and the exon sequence. In some respects, the intron sequence is completely excised from the RNA genetic construct (i.e., the precursor circRNA). In some respects, the resulting circRNA does not contain any intron sequence. In some respects, the resulting circRNA does not contain one or more intron sequence residues. In some respects, the resulting circRNA does not contain one or more nucleotide sequences derived from organisms derived from the 3' intron sequence, i.e., the resulting circRNA does not contain an "excision scar".

[0314] In some respects, precursor circRNAs can form circular RNAs. In some respects, precursor circRNAs can form circular RNAs in the presence of exogenous G. In some respects, precursor circRNAs can form circular RNAs where the circular RNA does not contain excision scars.

[0315] II.C.5. Exemplary Genetic Construct E In some respects, a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA) comprises the following formula: 5'-PBA-3S-E-3', wherein P contains a promoter (e.g., T7, T3, SP6, etc.), B contains a 5' intron sequence containing an internal guide sequence (IGS), A contains a 3' intron sequence, 3S contains a 3' splicing site, and E contains an exon sequence containing a catalytic nucleotide; and wherein the promoter (P) should be understood to be included in the DNA genetic construct but not in the RNA genetic construct.

[0316] In some aspects, the intron sequence comprises a nucleotide sequence derived from a group I intron. In some aspects, the group I intron comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 1 or 6. In some aspects, the group I intron consists of SEQ ID NO: 1 or 6.

[0317] In some aspects, the 5' intron sequence contains an internal guide site (IGS). In some aspects, the IGS contains a nucleotide sequence of CTTAA (or CUUAA) or TTGAG (or UUGAG). In some aspects, the 5' intron sequence contains a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 175 or 176. In some aspects, the 5' intron sequence consists of SEQ ID NO: 175 or 176.

[0318] In some respects, the 3' intron sequence contains a catalytic core. In other respects, the catalytic core contains AGAGA[N]. 6-41 A[N] 43-45 A sequence of GUCCs, where N is any one of A, U, C, or G, and where [N] 6-41 Or [N] 43-45 Each N in each chain can independently be any of A, U, C, or G. In some respects, the catalytic core is composed of AGAGA[N]. 6-41 A[N] 43-45 The GUCC structure is composed of N, where N is any one of A, U, C, or G, and [N] is... 6-41 Or [N] 43-45Each N in each strand can independently be any one of A, U, C, or G. In some aspects, the 3' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with any of SEQ ID NO: 177 or 178. In some aspects, the 3' intron sequence consists of any one of SEQ ID NO: 177 or 178.

[0319] In some aspects, the 5' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with any of SEQ ID NO: 175 or 176, and the 3' intron sequence comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with any of SEQ ID NO: 177 or 178. In some aspects, the 5' intron sequence consists of any of SEQ ID NO: 175 or 176, and the 3' intron sequence consists of any of SEQ ID NO: 177 or 178.

[0320] In some aspects, the 3' splice site comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 179 or 180. In some aspects, the 3' splice site consists of any one of SEQ ID NO: 179 or 180.

[0321] In some respects, the 5' intron sequence and the 3' intron sequence together are smaller than the complete intron.

[0322] In some respects, the 3' splice site is located at the boundary between the 3' intron sequence and the exon sequence. In some respects, the intron sequence is completely excised from the RNA genetic construct (i.e., the precursor circRNA). In some respects, the resulting circRNA does not contain any intron sequence. In some respects, the resulting circRNA does not contain one or more intron sequence residues. In some respects, the resulting circRNA does not contain one or more nucleotide sequences derived from organisms derived from the 3' intron sequence, i.e., the resulting circRNA does not contain an "excision scar".

[0323] In some respects, precursor circRNAs can form circular RNAs. In some respects, precursor circRNAs can form circular RNAs in the presence of exogenous G. In some respects, precursor circRNAs can form circular RNAs where the circular RNA does not contain excision scars.

[0324] In some aspects, the genetic constructs disclosed herein comprise a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 113-122, wherein “N” represents any one of A, T (or U), C, or G; and wherein “n” represents any integer. In some aspects, “(N)n” comprises the nucleotide sequence of the exon sequence. In some aspects, “(N)n” comprises the nucleotide sequence of the exon sequence and the nucleotide sequence of IRES. Non-limiting examples of the constructs disclosed herein are shown in Table 13.

[0325] In some aspects, the genetic constructs disclosed herein comprise nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 123-132, wherein “N” represents any one of A, T (or U), C, or G; and wherein “n” represents any integer. In some aspects, “(N)n” comprises the nucleotide sequence of the exon sequence. In some aspects, “(N)n” comprises the nucleotide sequence of the exon sequence and the nucleotide sequence of IRES.

[0326] In some aspects, the RNA transcript comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 123-132, wherein “N” represents any one of A, T (or U), C, or G; and wherein “n” represents any integer transcribed from the DNA genetic construct disclosed herein. In some aspects, “(N)n” comprises the nucleotide sequence of the exon sequence. In some aspects, “(N)n” comprises the nucleotide sequence of the exon sequence and the nucleotide sequence of IRES.

[0327] Non-limiting examples of the constructs of the present invention and RNA transcripts transcribed from the DNA genetic constructs disclosed herein are shown in Table 14.

[0328] Table 14. Exemplary genetic constructs and exemplary RNA transcripts transcribed from DNA genetic constructs disclosed herein ((N)n is the position of the exon sequence)

[0329] III. Carrier In some respects, the genetic construct, either DNA or RNA, is contained within the vector. In some respects, the vector is a DNA vector. In some respects, the vector is an RNA vector. In some respects, the vector is a DNA / RNA hybrid vector. In some respects, the vector is a plasmid.

[0330] In some aspects, the vector is produced by any of the methods known in the art for producing a vector containing a genetic construct. In some aspects, the vector is produced by chemical synthesis. In some aspects, the vector is produced by molecular cloning. In some aspects, the vector comprises naturally occurring nucleosides, naturally occurring sugar moieties, naturally occurring phosphate groups, naturally occurring nucleic acid backbones, naturally occurring nucleosides, naturally occurring nucleoside linkages, naturally occurring nucleotides, modified nucleosides, modified sugar moieties, modified phosphate groups, modified nucleic acid backbones, modified nucleosides, modified nucleoside linkages, modified nucleotides, or any combination thereof. In some aspects, the genetic construct or vector is suitable for in vivo transcription. In some aspects, the vector is suitable for in vitro transcription. In some aspects, the vector is suitable for both in vivo and in vitro transcription.

[0331] In some respects, the genetic constructs described herein (e.g., contained in a vector) contain one or more additional components that contribute to the function of the DNA genetic construct, the precursor circRNA, or the circRNA produced as disclosed herein.

[0332] In some aspects, the genetic constructs disclosed herein include one or more additional components, wherein the one or more additional components, for example, promote transcription of precursor circRNA from the DNA genetic construct, promote circularization of precursor circRNA, enhance expression of the encoded payload, increase the stability of the payload sequence, or any combination thereof.

[0333] Non-limiting examples of additional components that may be used in this disclosure include: (1) an internal ribosome entry site (IRES), (2) an untranslated region (UTR), (3) a sequence encoding a signal peptide, (4) a translation initiation sequence, (5) a sequence encoding an RNA-binding protein, (6) a sequence encoding a 2A peptide, (7) a translation enhancer element, or (8) any combination of (1) to (7).

[0334] In some respects, the vectors disclosed herein also contain additional genetic elements. These additional genetic elements include: restriction endonuclease recognition sites, selectivity markers, antibiotic resistance-conferring sequences (e.g., ampicillin or kanamycin resistance elements), self-cleavage site coding sequences (e.g., self-cleaving 2A peptides: P2A, T2A, and F2A with or without furin cleavage sites), translation enhancer elements (TEEs), origin of replication sites, and promoter sequences (e.g., T7, T3, or Sp6 phage polymerase promoters).

[0335] In some respects, the vectors disclosed herein contain translation enhancer elements (TEEs). As used herein, the term "translation enhancer element" refers to a cis-acting sequence that increases the expression of a protein encoded by a nucleotide sequence. Non-limiting examples of TEEs that may be used with this disclosure are known in the art, see, for example, US20130177581A, which is incorporated herein by reference in its entirety. When such a precursor circRNA or circRNA as disclosed herein is introduced into target cells, for example, expression of the payload is increased compared to a corresponding precursor circRNA or circRNA in which the payload sequence does not contain a TEE.

[0336] In some respects, the TEE is located between the transcription promoter and the start codon of a sequence (e.g., the payload sequence). In some respects, the TEEs available in this disclosure have at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity with any TEE provided in the following patents: US Publication No. US US20140147454, US20090226470, US20070048776, US20130177581, US20110124100, WO1999024595, WO2012009644, WO2009075886, WO2007025008, US Patent No. 6,310,197, US Patent No. 6,849,405, US Patent No. 7,456,273, and US Patent No. 7,183,395, each of which is incorporated herein by reference in its entirety.

[0337] In some respects, the vectors disclosed herein contain multiple TEEs. For example, in some respects, the precursor circRNA or circRNA generated as disclosed herein contains a payload sequence, wherein the payload sequence contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or more than 60 TEE sequences. In some respects, the TEE sequences in the 5' UTR of the RNA (e.g., modified RNA) are the same or different TEE sequences. In some respects, TEE sequences are in patterns repeated once, twice, or more than three times, such as ABABAB, AABBBAABBAABB, or ABCABCABC, or variations thereof. In these patterns, each letter A, B, or C represents a different TEE sequence at the nucleotide level.

[0338] In some aspects, the vectors disclosed herein contain an antibiotic resistance-contributing sequence. In some aspects, the genetic constructs disclosed herein contain at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical antibiotic resistance-contributing sequence to the antibiotic resistance-contributing sequence of SEQ ID NO: 111. In some aspects, the genetic constructs disclosed herein (e.g., contained in a vector), precursor RNA, or circular RNA contains the antibiotic resistance-contributing sequence of SEQ ID NO: 111.

[0339] In some respects, the DNA genetic constructs disclosed herein (e.g., contained in a vector), precursor circRNAs, and circRNAs have lengths of 300 to 10,000, 400 to 9,000, 500 to 8,000, 600 to 7,000, 700 to 6,000, 800 to 5,000, 900 to 5,000, 1,000 to 5,000, 1,100 to 5,000, 1,200 to 5,000, 1,300 to 5,000, 1,400 to 5,000, and / or 1,500 to 5,000 nucleotides. In some respects, the DNA genetic constructs disclosed herein (e.g., contained in a vector), precursor circRNAs, and circRNAs are at least 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, or 5000 nt. In some respects, the DNA genetic constructs disclosed herein (e.g., contained in a vector), precursor circRNAs, and circRNAs are no longer than 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt. In some implementations, the DNA, linear RNA, and / or circular RNA polynucleotides provided herein are approximately 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length.

[0340] IV. Modified nucleotides In some aspects, the genetic constructs disclosed herein contain one or more modified nucleosides. In some aspects, one or more modified nucleosides include 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudo-uridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudo-uridine, inosine, α-thio-guanosine, 8- Oxo-guanosine, O6-methyl-guanosine, 7-deazo-guanosine, N1-methyladenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deazo-adenosine, pyrrole-cytidine, 5-methyl-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5-iodo-cytidine, and combinations thereof.

[0341] In some respects, the genetic constructs disclosed herein contain one or more uridines that have been substituted with modified nucleosides. In some respects, the modified nucleosides that replace uridines are pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0342] V. Circular RNA In some respects, circRNAs prepared using the precursor circRNA constructs or methods described herein exhibit greater stability than linear RNAs (e.g., mRNAs) containing the same sequence. In some respects, the stability of circRNAs prepared using the precursor circRNA constructs or methods described herein is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times greater than that of linear RNAs (e.g., mRNAs) containing the same sequence. In some respects, circRNAs prepared using the precursor circRNA constructs or methods described herein exhibit greater stability than linear RNAs (e.g., mRNAs) containing the same sequence when applied to cells. In some respects, cells are contained within organisms. In some respects, organisms are humans. In some respects, circRNAs prepared by the precursor circRNA constructs or methods described herein exhibit greater stability than linear RNAs (e.g., mRNAs) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail, and encoding the same payload. In some respects, the stability of circRNAs prepared using the precursor circRNA constructs or methods described herein is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times greater than that of linear RNAs (e.g., mRNAs) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail, and encoding the same protein. In some respects, when applied to cells, circRNAs prepared using the precursor circRNA constructs or methods described herein exhibit greater stability than linear RNAs (e.g., mRNAs) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail, and encoding the same protein. In some respects, cells are contained within organisms. In some respects, the organism is a human being. In some respects, the circRNA prepared by the precursor circRNA constructs or methods described herein has a stability greater than or equal to a predetermined threshold. In some respects, the predetermined threshold is the stability of a reference linear RNA (e.g., mRNA) containing the same sequence as the circular RNA.In some respects, the predetermined threshold is the stability of a reference linear RNA (e.g., mRNA) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail, and encoding the same payload. In some respects, the circRNA exhibits stability greater than or equal to the predetermined threshold when applied to cells. In some respects, cells are contained within an organism. In some respects, the organism is a human being.

[0343] In some respects, the circRNAs prepared by the precursor circRNA constructs or methods described herein have a longer half-life or functional half-life than linear RNAs (e.g., mRNAs) containing the same sequence. In some respects, the half-life or functional half-life of the circRNAs prepared by the precursor circRNA constructs or methods described herein is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times longer than the half-life or functional half-life of linear RNAs (e.g., mRNAs) containing the same sequence. In some respects, when applied to cells, circRNAs prepared using the precursor circRNA constructs or methods described herein have a longer half-life or functional half-life than linear RNAs (e.g., mRNAs) containing the same sequence. In some respects, cells are contained within an organism. In some respects, the organism is a human. In some respects, circRNAs prepared using the precursor circRNA constructs or methods described herein have a longer half-life or functional half-life than linear RNAs (e.g., mRNAs) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail, and encoding the same payload. In some respects, the half-life or functional half-life of circRNAs prepared by the precursor circRNA constructs or methods described herein is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times greater than the half-life or functional half-life of linear RNAs (e.g., mRNAs) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail and encoding the same payload. In some respects, when applied to cells, the circRNA prepared by the precursor circRNA construct or method described herein has a longer half-life or functional half-life than a linear RNA (e.g., mRNA) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail and encoding the same payload. In some respects, cells are contained in an organism. In some respects, the organism is a human. In some respects, the circRNA has a half-life or functional half-life in cells that is greater than or equal to a predetermined threshold. In some respects, the predetermined threshold is the half-life or functional half-life of a reference linear RNA (e.g., mRNA) containing the same sequence as the circRNA.In some respects, the predetermined threshold is the half-life or functional half-life of a reference linear RNA (e.g., mRNA) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail, and encoding the same payload. In some respects, when administered to cells, the circRNA has a half-life or functional half-life greater than or equal to the predetermined threshold. In some respects, cells are contained within an organism. In some respects, the organism is a human being.

[0344] In some aspects, the circRNAs prepared using the precursor circRNA constructs or methods described herein have a half-life or functional half-life of at least 5 hours, 10 hours, 15 hours, 20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, or 80 hours. In some aspects, the circular RNAs provided herein have a half-life of 5-80, 10-70, 15-60, and / or 20-50 hours. In some aspects, the circRNAs prepared using the precursor circRNA constructs or methods described herein have a half-life or functional half-life of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In some aspects, the half-life or functional half-life is determined by nucleic acid assay. In some aspects, the functional half-life is determined by functional protein assay. In some aspects, the circular RNA contains a nucleotide sequence encoding a reporter gene. In some aspects, the functional half-life is determined by an in vitro luciferase assay measuring the expression of the reporter gene. In some respects, the functional half-life is determined by an in vitro luciferase assay, wherein the activity of Gaussian luciferase (GLuc) is measured in cells expressing circular RNA polynucleotides (e.g., HepG2) at 1, 2, 6, 12, or 24 hours every 1, 2, 3, 4, 5, 6, 7, or 14 days. In some respects, the functional half-life is determined by an in vivo assay, wherein fluorescence (e.g., from GFP protein) is measured in cells expressing circular RNA (e.g., HepG2) at 1, 2, 6, 12, or 24 hours every 1, 2, 3, 4, 5, 6, 7, or 14 days. In other respects, the functional half-life is determined by an in vivo assay, wherein the level of the protein encoded by the expressed sequence of the circular RNA polynucleotide is measured in a patient serum or tissue sample at 1, 2, 6, 12, or 24 hours every 1, 2, 3, 4, 5, 6, 7, or 14 days.

[0345] In some respects, circRNAs prepared using the precursor circRNA constructs or methods described herein exhibit a larger expression level than linear RNAs (e.g., mRNAs) containing the same sequence. In some respects, the expression level of circRNAs prepared using the precursor circRNA constructs or methods described herein is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times greater than that of linear RNAs (e.g., mRNAs) containing the same sequence. In some respects, when applied to cells, circRNAs prepared using the precursor circRNA constructs or methods described herein exhibit a larger expression level than linear RNAs (e.g., mRNAs) containing the same sequence. In some respects, cells are contained within organisms. In some respects, an organism is a human being. In some respects, circRNAs prepared by the precursor circRNA constructs or methods described herein have a much larger expression level than linear RNAs (e.g., mRNAs) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail and encoding the same payload. In some respects, the expression levels of circRNAs prepared by the precursor circRNA constructs or methods described herein are at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times higher than the expression levels of linear RNAs (e.g., mRNAs) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail and encoding the same payload. In some respects, when applied to cells, the circRNA prepared by the precursor circRNA constructs or methods described herein has a larger expression level than a linear RNA (e.g., mRNA) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail and encoding the same payload. In some respects, cells are contained in an organism. In some respects, the organism is a human. In some respects, the circRNA has an expression level greater than or equal to a predetermined threshold. In some respects, the predetermined threshold is the expression level of a reference linear RNA (e.g., mRNA) containing the same sequence as the circRNA.In some respects, the predetermined threshold is the expression level of a reference linear RNA (e.g., mRNA) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail, and encoding the same payload. In some respects, the circRNA has an expression level greater than or equal to the predetermined threshold when applied to cells. In some respects, cells are contained within an organism. In some respects, the organism is a human being.

[0346] In some respects, circRNAs prepared by the precursor circRNA constructs or methods described herein have lower immunogenicity than linear RNAs (e.g., mRNAs) containing the same sequence. In some respects, the immunogenicity of the circRNAs described herein is at most 1 / 0.5, 1 / 1, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 100, 1 / 150, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000 of the immunogenicity of linear RNAs (e.g., mRNAs) containing the same sequence. In some respects, the circular RNAs described herein exhibit lower immunogenicity when applied to cells than linear RNAs (e.g., mRNAs) containing the same sequence. In some respects, cells are contained within an organism. In some respects, the organism is a human. In some respects, the circRNAs provided herein exhibit lower immunogenicity than linear RNAs (e.g., mRNAs) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail, and encoding the same payload. In some respects, the immunogenicity of the circRNAs provided herein is that of linear RNAs containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail, and encoding the same payload. The immunogenicity of (e.g., mRNA) is at most 1 / 0.5, 1 / 1, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 100, 1 / 150, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000. In some respects, when administered to cells, the circRNAs provided herein exhibit lower immunogenicity than linear RNAs (e.g., mRNAs) containing the same sequence, modifications, optimized UTRs, caps, and / or polyA tails and encoding the same payload. In some respects, cells are contained within an organism. In some respects, the organism is a human. In some respects, the circRNAs exhibit immunogenicity below or equal to a predetermined threshold. In some respects, the predetermined threshold is the immunogenicity of a reference linear RNA (e.g., mRNA) containing the same sequence as the circRNA.In some respects, the predetermined threshold is the immunogenicity of a reference linear RNA (e.g., mRNA) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail, and encoding the same payload. In some respects, the circRNA has immunogenicity below or equal to the predetermined threshold when applied to cells. In some respects, cells are contained within an organism. In some respects, the organism is a human being.

[0347] In some respects, when exposed to an organism's immune system or certain types of immune cells, the circular RNAs described herein exhibit lower immunogenicity than equivalent linear RNAs (i.e., linear RNAs containing the same sequence as circular RNAs, or linear RNAs (e.g., mRNAs) containing the same sequence, the same modifications, the same optimized UTR, the same cap and / or polyA tail as circular RNAs and encoding the same protein).

[0348] In some respects, the circRNAs described herein do not contain excision scars and have lower immunogenicity than the same circRNAs that contain excision scars and contain the same sequence. In some respects, the immunogenicity of the circRNAs provided herein is at most 1 / 0.5, 1 / 1, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 100, 1 / 150, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000 of the immunogenicity of the same circRNA containing the excision scar and containing the same sequence. In some respects, the circRNAs provided herein exhibit lower immunogenicity when applied to cells than the same circRNAs containing excision scars and containing the same sequence. In some respects, cells are contained within an organism. In some respects, the organism is a human. In some respects, the circRNAs provided herein exhibit lower immunogenicity than the same circRNAs containing excision scars and containing the same sequence, the same modifications, the same optimized UTR, and encoding the same payload. In some respects, the immunogenicity of the circRNAs provided in this article is at most 1 / 0.5, 1 / 1, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 100, 1 / 150, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000 of the immunogenicity of the same circRNAs containing excision scars and containing the same sequence, the same modifications, the same optimized UTRs and encoding the same payload. In some respects, when applied to cells, the circRNAs provided herein exhibit lower immunogenicity than identical circRNAs containing excision scars and containing the same sequence, modifications, optimized UTRs, and encoding the same payload. In some respects, cells are contained within an organism. In some respects, the organism is a human. In some respects, the circRNAs exhibit immunogenicity below or equal to a predetermined threshold. In some respects, the predetermined threshold is the immunogenicity of a reference circRNA containing excision scars and containing the same sequence as the circRNA.In some respects, the predetermined threshold is the immunogenicity of a reference circRNA that includes the excised scar and contains the same sequence, the same modifications, the same optimized UTR, and encodes the same payload. In some respects, the circRNA has immunogenicity below or equal to the predetermined threshold when applied to cells. In some respects, cells are contained within an organism. In some respects, the organism is a human being.

[0349] In some respects, the circular RNAs described herein have lower immunogenicity than their equivalent circRNAs containing excision scars (i.e., circRNAs containing excision scars and containing the same sequence, the same modifications, the same optimized UTR and encoding the same payload) when exposed to the organism's immune system or certain types of immune cells.

[0350] In some respects, the circRNAs described herein are associated with the regulation of cytokines when exposed to the immune system or certain types of immune cells of an organism. Specifically, when exposed to the immune system or certain types of immune cells of an organism, the circRNAs described herein are associated with reductions in IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and / or TNFα compared to equivalent linear RNAs (i.e., linear RNAs containing the same sequence as the circRNA, or linear RNAs containing the same sequence, modifications, optimized UTRs, caps, and / or polyA tails and encoding the same payload as circular RNAs (e.g., mRNA)).

[0351] In some respects, the circRNAs provided herein do not contain excision scars. In some respects, the circRNAs provided herein are associated with the regulation of cytokines when exposed to the immune system or certain types of immune cells of an organism. In some respects, when exposed to the immune system or certain types of immune cells of an organism, the circRNAs provided herein are associated with reductions in IFN-β1, RIG-I, IL-2, IL-6, IFNy, and / or TNFα compared to equivalent circRNAs containing excision scars (i.e., circRNAs containing the same sequence as the circRNA and also containing excision scars; or circRNAs containing the same sequence, the same modifications, the same optimized UTR and encoding the same payload and also containing excision scars (e.g., mRNA)).

[0352] In some respects, when exposed to an organism’s immune system or certain types of immune cells, the circular RNAs presented herein are associated with less induction of IFN-β1, RIG-I, IL-2, IL-6, IFNy, and / or TNFα transcripts compared to equivalent linear RNAs (i.e., linear RNAs containing the same expression sequence as circular RNAs, or linear RNAs containing the same expression sequence, the same modifications, optimized UTRs, caps, and / or polyA tails as circular RNAs and encoding the same proteins (e.g., mRNAs)).

[0353] In some respects, when exposed to an organism’s immune system or certain types of immune cells, the circular RNAs presented herein are associated with less induction of IFN-β1, RIG-I, IL-2, IL-6, IFNy, and / or TNFα transcripts when exposed to an organism’s immune system or certain types of immune cells. These circular RNAs contain equivalent circRNAs containing excision scars (i.e., circRNAs containing the same sequence as circRNAs and further containing excision scars; or circular RNAs containing the same sequence, the same modifications, the same optimized UTRs and encoding the same payload and further containing excision scars (e.g., mRNAs)).

[0354] In some aspects, transcription of precursor circRNA from a DNA genetic construct (e.g., contained in a vector) occurs in vitro. In some aspects, in vitro transcription of precursor circRNA from a DNA genetic construct (e.g., contained in a vector) occurs in cells. In some aspects, the cells are prokaryotic cells. In some aspects, the cells are eukaryotic cells. In vitro transcription of precursor RNA from a genetic construct (e.g., contained in a vector) in cells can be performed via an added polymerase or a polymerase encoded by nucleic acids transfected into the cell, or via an endogenous polymerase (e.g., RNA polymerase II). In some aspects, precursor circRNA is transcribed in the cytoplasm of the cell. In some aspects, precursor circRNA is transcribed in the nucleus of the cell. In some aspects, in vitro transcription of precursor circRNA from a DNA genetic construct (e.g., contained in a vector) occurs in a cell-free reaction. In cell-free reactions, in vitro transcription of precursor RNA from a genetic construct (e.g., contained in a vector) can be performed by any RNA polymerase. In some aspects, the RNA polymerase is a bacteriophage RNA polymerase (e.g., T7, T3, or Sp6).

[0355] In some respects, the circularization of precursor circRNAs that produce circRNAs occurs in vitro. In some respects, the in vitro circularization of precursor circRNAs that produce circRNAs occurs in cells. In some respects, the cells are prokaryotic cells. In some respects, the cells are eukaryotic cells. In some respects, circRNAs are produced in the cytoplasm of the cell. In some respects, circRNAs are produced in the nucleus of the cell. In some respects, the circularization of precursor circRNAs that produce circRNAs occurs in cell-free reactions.

[0356] In some aspects, transcription of precursor circRNA from a DNA genetic construct (e.g., contained in a vector) occurs in vivo. In other aspects, a DNA genetic construct (e.g., contained in a vector) is applied to cells contained in a multicellular organism, and the precursor circRNA is transcribed from the DNA genetic construct (e.g., contained in a vector) in the cells contained in the multicellular organism.

[0357] In some aspects, the circularization of the precursor circRNA that produces the circRNA occurs in vivo. In some aspects, a DNA genetic construct (e.g., contained in a vector) is applied to cells contained in a multicellular organism, the precursor circRNA is transcribed from the DNA genetic construct (e.g., contained in a vector), and the precursor circRNA is circularized in the cells contained in the multicellular organism. In some aspects, the precursor circRNA is applied to cells contained in a multicellular organism, and the precursor circRNA is circularized in the cells contained in the multicellular organism.

[0358] In some respects, the circRNAs provided herein are applied to cells. In some respects, sequences encoding payloads contained in the circular RNAs described herein are expressed in cells. In some respects, cells are contained in organisms. In some respects, the organism is an animal. In some respects, the animal is a human.

[0359] VI. Precursor circRNA circularization In some respects, the precursor circRNA contains both a 5' and a 3' splice site. In these respects, the cyclization of the precursor circRNA occurs via a two-step transesterification reaction with exogenous GTP (αG), whose 3'–OH acts as the initiating nucleophile. The binding of αG to the G-binding site in P7 positions the 3'–OH of GTP to attack the 5' splice site. During the first transesterification step, αG is linked to the 5' end of the intron RNA via a 3'-5' phosphodiester bond. This step is followed by a conformational change that allows the terminal 3' guanosine (ωG) of the upstream exon to exchange position with αG and occupy the G-binding site, initiating the second transesterification reaction. The 3'–OH of the upstream exon attacks the 3' splice site (an interaction facilitated by the formation of P10), promoting the ligation of the upstream and downstream exons and the release of the intron RNA.

[0360] In some respects, the precursor circRNA contains a 3' splice site and a catalytic nucleotide. In these respects, the cyclization of the precursor circRNA occurs via a one-step transesterification reaction, in which the terminal 3' guanosine (ωG) of the upstream exon exchanges position with αG and occupies the G-binding site to initiate the transesterification reaction. The 3'–OH of the upstream exon attacks the 3' splice site (an interaction promoted by the formation of P10), facilitating the joining of upstream and downstream exons and the release of the intron RNA.

[0361] VII. Compositions containing genetic constructs and methods for preparing circRNA In some respects, this document provides compositions comprising a genetic construct capable of forming the circular RNA disclosed herein (i.e., a precursor circRNA or a DNA genetic construct capable of transcribing a precursor circRNA) and one or more additional components.

[0362] In some aspects, the additional component is one or more metal ions. In some aspects, one or more metal ions may be used in compositions comprising the precursor circRNA disclosed herein to produce circRNA. In some aspects, the metal ion is a cation. In some aspects, the cation is a monovalent cation. In some aspects, the monovalent cation is Na+, K+, NH4+, which may be included in the form of NaCl, KCl, NH4Cl, ammonium acetate, and ammonium sulfate. In some aspects, the cation is a divalent cation. In some aspects, the metal ion is Mg2+, Ca2+, Cu2+, Mn2+, Zn2+, Pb2+, Co2+, Cd2+, Sn2+, Ni2+, Fe2+, Ba2+, or Sr2+. In some aspects, the cation is a trivalent cation. In some aspects, the metal ion is ytterbium (Yb3+).

[0363] In some aspects, the additional component is RNA polymerase. In some aspects, the RNA polymerase can be used in compositions comprising the DNA genetic constructs disclosed herein to produce the precursor circRNA disclosed herein. In some aspects, the RNA polymerase is T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or any combination thereof.

[0364] In some respects, the additional components are salts, buffers, or any additional components that can facilitate the transcription of the DNA genetic constructs disclosed herein into the precursor circRNAs disclosed herein, and / or any additional components that can facilitate the circularization of the precursor circRNAs disclosed herein to produce the circRNAs disclosed herein.

[0365] Any suitable method known in the published literature can be used to transcribe the DNA genetic constructs disclosed herein into the precursor circRNAs disclosed herein, and / or circularize the precursor circRNAs disclosed herein to produce the circRNAs disclosed herein.

[0366] The DNA genetic constructs disclosed herein can be transcribed into the precursor circRNAs disclosed herein in vitro, in vivo, or in any combination thereof. Similarly, the precursor circRNAs disclosed herein in vitro or in vivo can be circularized to produce the circRNAs disclosed herein in vitro, in vivo, or in any combination thereof.

[0367] Vectors containing the DNA genetic constructs and precursor circRNAs provided herein can be prepared using standard techniques in molecular biology. For example, the various elements of the vectors provided herein can be obtained through chemical synthesis or recombinant methods. For example, the genetic elements contained in the genetic constructs can be derived from cDNA (derived from cells) or from vectors known to contain the genetic elements of interest. Alternatively, the genetic elements of the genetic constructs provided herein can also be synthesized based on known sequences rather than cloned. The complete sequence can be assembled from overlapping oligonucleotides prepared by standard methods. See, for example, Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; and Jay et al., J. Biol. Chem. (1984) 259:6311. Specific nucleotide sequences can be obtained from vectors carrying the desired sequence or synthesized completely or partially using various oligonucleotide synthesis techniques known in the art. In addition, nucleotide sequences can be modified or amplified using any techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques when appropriate. One method to obtain the nucleotide sequence encoding the desired vector element is by annealing multiple sets of complementary overlapping synthetic oligonucleotides produced in a conventional automated polynucleotide synthesizer, followed by ligation with a suitable DNA ligase and amplification of the ligated nucleotide sequence via PCR. See, for example, Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. Alternatively, oligonucleotide directed synthesis (Jones et al., Nature (1986) 54:75-82), oligonucleotide directed mutagenesis of pre-existing nucleotide regions (Riechmann et al., Nature (1988) 332:323-327 and Verhoeyen et al., Science (1988) 239: 1534-1536), and enzymatic filling of nicked oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86: 10029-10033).

[0368] The precursor circRNAs provided herein can be generated by incubating the vectors provided herein under conditions that allow transcription of the precursor RNA encoded by the vector. In some aspects, the precursor RNAs are synthesized by incubating the genetic constructs (e.g., contained in the vector) containing an RNA polymerase promoter provided herein under conditions that allow in vitro transcription with a compatible RNA polymerase (e.g., T7, T3, or Sp6 RNA polymerase). In some aspects, the genetic constructs (e.g., contained in the vector) are incubated in cells and transcribed by phage RNA polymerase or by RNA polymerase II.

[0369] In some respects, this article provides a method for generating precursor circRNA by in vitro transcription using a DNA genetic construct (e.g., contained in a vector) provided herein as a template (e.g., a vector containing an RNA polymerase promoter provided herein). In some respects, the resulting precursor circRNA can be used to generate the circRNA provided herein by incubating the resulting precursor circRNA at a temperature (e.g., between 20°C and 60°C) in the presence of magnesium and ammonium ions.

[0370] In some aspects, the composition containing circular RNA is purified. The circular RNA can be purified by any known method in the art, such as column chromatography, gel filtration chromatography, and size exclusion chromatography. In some aspects, purification includes one or more of the following steps: phosphatase treatment, HPLC size exclusion purification, and RNase R digestion. In some aspects, purification sequentially includes the following steps: RNase R digestion, phosphatase treatment, and HPLC size exclusion purification. In some aspects, purification includes reversed-phase HPLC. In some aspects, the purified composition contains less double-stranded RNA, DNA clips, triphosphorylated RNA, phosphatase proteins, protein ligases, capping enzymes, and / or nicked RNA than the unpurified RNA. In some aspects, the purified composition has lower immunogenicity than the unpurified composition. In some aspects, immune cells exposed to the purified composition produce less IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and / or TNFα than immune cells exposed to the unpurified composition.

[0371] VIII. Pharmaceutical Compositions In some aspects, this document provides pharmaceutical compositions comprising a DNA genetic construct disclosed herein (e.g., contained in a vector), a precursor circRNA, or circRNA. In some aspects, the DNA genetic construct disclosed herein (e.g., contained in a vector), the precursor circRNA, or circRNA may be used as a therapeutic agent. In some aspects, the therapeutic agent is a DNA genetic construct provided herein (e.g., contained in a vector). In some aspects, the therapeutic agent is a precursor circRNA provided herein. In some aspects, the therapeutic agent is a circRNA provided herein. In some aspects, the therapeutic agent is a vector containing a genetic construct provided herein. In some aspects, the therapeutic agent is a cell (e.g., human cells, such as human T cells) containing a genetic construct provided herein. In some aspects, the composition also comprises a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, buffer, excipient, salt, or stabilizer in the form of a lyophilized formulation or an aqueous solution, see, for example, Remington: The Science and Practice of Pharmacy, 20th edition (2000), edited by Lippincott Williams and Wilkoins, KE Hoover. As used herein, “acceptable” means that the carrier must be compatible with the active ingredient of the composition and harmless to the subject to be treated. In some respects, the carrier can stabilize the active ingredient.

[0372] In some aspects, the pharmaceutical composition can be formulated for intratumoral, intrathecal, intravenous, subcutaneous, inhalation, intradermal, intralymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, intranasal, percutaneous, sublingual, submucosal, transdermal, or transmucosal administration. In some aspects of this disclosure, the pharmaceutical composition can be formulated for intratumoral injection. As used herein, intratumoral injection refers to direct injection into the tumor. High concentrations of the composition can be obtained in situ while using small amounts of the drug. Local delivery of immunotherapy allows for multiple combination therapies while preventing significant systemic exposure and off-target toxicity.

[0373] In some respects, the pharmaceutical composition can be formulated for intramuscular, intravenous, or subcutaneous injection.

[0374] IX. General Techniques Unless otherwise stated, the practice of this disclosure will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (edited by MJ Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JE Cellis, 1998) Academic Press; Animal Cell Culture (edited by RI Freshney, 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, JB Giffiths, and DG Newell, 1993-8) J. Wiley and Sons; Method of Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (edited by DM Weir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (edited by JM Miller and MPCalos, 1987); Current Protocols in Molecular Biology (edited by FM Ausubel et al., 1987): PCR: The Polymerase Chain Reaction (edited by Mullis et al., 1994); Current Protocols in Immunology (edited by JE Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (edited by CA Janeway and P.).Travers (1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (edited by D. Catty, IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanette and JD Capra, Harwood Academic Publishers, 1995). Without further elaboration, it is believed that those skilled in the art will be able to make the most of this disclosure based on the foregoing description. All publications cited herein (including those listed above and elsewhere in this disclosure) are incorporated herein by reference in their entirety.

[0375] Table 15: Sequence List ( nnnnnnnnnn Exemplary locations indicating exon sequences; mmmmmmmmmm indicates exemplary locations of IRES.

[0376] Example The following experiments (Examples 1-4) describe the precursor circRNA construct and the method for preparing circRNA using the precursor circRNA construct. Non-limiting examples of precursor circRNA constructs include, but are not limited to: Figures 1A to 1E The example constructor in the example.

[0377] Example 1: Generation of DNA genetic constructs DNA genetic constructs containing the 3' intron sequences and 5' intron sequences of SEQ ID NO 1-5 were chemically synthesized in the form of double-stranded DNA.

[0378] The genetic construct was cloned into a DNA vector (pUC plasmid vector). The genetic construct contains SEQ ID NO: 42-51. The introns undergo catalytic splicing, which removes the 3' and 5' intron sequences from the transcribed RNA sequence and circularizes the exon sequences held by the introns.

[0379] The mechanism of cyclization reaction is as follows: 1) Free exogenous guanosine (GTP) binds to the catalytic core in the rearranged 3' intron. 2) Guanosine performs nucleophilic attack on the 5' splicing site of rearranged 5' introns, cutting the introns out of the exon sequence, and 3) The 3'-OH group of uracil at the 5' splice site then performs nucleophilic attack on the phosphate group between the intron and ω-guanosine (terminal guanosine in the exon sequence) at the 3' splice site, cutting the 3' intron out of the sequence and joining the exon ends together to form a circular RNA. Figure 2 ).

[0380] The introns contained within the generated genetic construct are group I introns. The 5' splice site, 3' splice site, and catalytic core all participate in coordinating the sequential cleavage-ligation reaction of group I introns. Exogenous G first binds to the catalytic core (also called the G site), forming a base triplet with the intron. Magnesium ions help coordinate the nucleophilic attack on the phosphate backbone at the 5' splice site and subsequently at the 3' splice site. The 5' splice site is located in a region called the P1 helix or P1 domain. Intron and exon sequences pair base-to-base in this region, producing the specificity required for proper cleavage at the 5' splice site. The intron component is called the internal guide sequence (IGS)—complementary to the exon sequence at 5 bp and to uracil at the 5' splice site. The IGS sequence is the exon sequence complementary to the IGS.

[0381] The group I introns contained in the genetic constructs disclosed herein are tRNAs derived from *Anabaena macrantha*. LEU(SEQ ID NO: 4), tRNA of *Vibrio zonatogenes* ILE (SEQ ID NO: 3) or modified T4 phage td (SEQ ID NO: 5). Anabaena PCC7120 (SEQ ID NO: 1) is used as a control.

[0382] The 3' and 5' intron sequences of group I introns were cloned into a plasmid vector. Figure 3A (See Figure 3D), with the exon sequence containing the 5' spacer sequence, 5' polyA sequence, IRES, GFP-encoded nucleotide sequence, 3' polyA sequence and 3' spacer sequence.

[0383] Example 2: Cycloning efficiency Circulation efficiency was determined by gel electrophoresis. Due to their different spatial conformations and molecular weights, linear and circular RNAs could be identified as distinct bands on the electrophoretic gel. Transcribed RNA was measured before and after circularization in the presence of GTP. (This is in contrast to RNA derived from Anabaena.) PCC7120 Compared to genetic constructs containing intron sequences derived from *Anabaena*, *Vibrio*, or modified T4 phage td introns, genetic constructs exhibited higher circularization efficiency. Furthermore, compared to constructs containing intron sequences derived from *Anabaena*... PCC7120 Compared to constructs with rearranged intron sequences, constructs containing intron sequences derived from *Azolla*, *Vibrio*, or modified T4 phage td introns showed reduced formation of polymorphs (Table 16 and 17). Figure 4 , Figures 5A to 5D ).

[0384] Table 16. Cycloning Efficiency Example 3: Expression efficiency of circular RNA To evaluate the expression efficiency of the generated circular RNA, a mixture of bacteria derived from *Vibrio zoea*, *Anabaena spp.*, modified T4 phage Td, and *Anabaena spp.* was used. PCC7120 Circulated RNA synthesized from the genetic construct containing the intron sequence was transfected into HEK293T cells. GFP expression was assessed by flow cytometry. The gene was derived from *Vibrio zoea*, *Anabaena spp.*, modified T4 phage Td, and *Anabaena spp.* PCC7120 The circular RNAs of the genetic constructs containing the intron sequences all showed considerable GFP expression levels. Figure 6 ).

[0385] These data demonstrate the feasibility of synthesizing circular RNAs with intron sequences derived from *Vibrio zoea*, *Anabaena*, and T4 phage Td, which have not been previously used in this area. Furthermore, compared to those derived from known *Anabaena* species... PCC7120 Compared to the intron sequences of *Vibrio azotoxinus*, *Azolla fusiforme*, and T4 phage Td, intron sequences showed advantages. Intron sequences of rearranged introns derived from *Vibrio azotoxinus*, *Azolla fusiforme*, and T4 phage Td exhibited increased transcription and circularization efficiency, as well as reduced polysynthesis formation.

[0386] It should be understood that the detailed description section, rather than the summary and abstract section, is intended to interpret the claims. The summary and abstract section may illustrate one or more, but not all, exemplary aspects of this disclosure as conceived by the inventors, and therefore is not intended to limit this disclosure and the appended claims in any way.

[0387] This disclosure has been described above using functional structural units that illustrate the implementation of specific functions and their relationships. For ease of description, the boundaries of these functional building blocks have been arbitrarily defined. Alternative boundaries may also be defined, provided that the specified functions and their interrelationships are properly performed.

[0388] The foregoing description of specific aspects will fully reveal the general characteristics of this disclosure, enabling others to readily modify and / or alter such specific aspects for various applications by applying knowledge in the art without excessive experimentation or departing from the general conception of this disclosure. Therefore, based on the teachings and guidance presented herein, such changes and modifications are intended within the meaning and scope of equivalents of the disclosed aspects. It should be understood that the wording or terminology herein is for descriptive and not limiting purposes, and that the terminology or terminology of this specification should be interpreted by a skilled person based on the teachings and guidance. The breadth and scope of this disclosure should not be limited by any of the foregoing exemplary aspects, but should be defined solely by the following claims and their equivalents.

Claims

1. A genetic construct comprising the following formula: 5'-HAEB-H'-3', Wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence (IGS), and H' contains a 3' homologous arm, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site, and wherein the genetic construct is capable of forming a circular RNA, the circular RNA not containing any exon sequences ("excision scar") derived from the genome from which the 5' intron sequence and the 3' intron sequence are derived.

2. The genetic construct of claim 1, wherein the 5' intron sequence and / or the 3' intron sequence are derived from group I introns.

3. The genetic construct of claim 1 or 2, wherein the 3' intron sequence and the 5' intron sequence together are smaller than the complete intron.

4. The genetic construct according to any one of claims 1 to 3, wherein the 5' intron sequence and / or the 3' intron sequence are derived from introns of: thymidine synthase (td) from T4 phage; or precursor tRNA from cyanobacteria such as *Anabaena spp.*, *Anabaena PCC7120*, *Nematocystis nigra* strain NIVA-CYA 142, or *Synechococcus*. LEU Precursor tRNA of nitrogen-fixing bacteria, Vibrio azotoxinus ILE ; 26S ribosomal RNA from Tetrahymena thermophila; from Papilionophora spp. COX2 Genes; or any combination thereof.

5. The genetic construct according to any one of claims 1 to 4, wherein the 5' intron sequence and / or the 3' intron sequence are derived from an intron of thymidine synthase (td) from T4 phage.

6. The genetic construct of any one of claims 1 to 5, wherein the 5' splice site comprises a site located between two nucleotides comprising U and N, wherein N is any nucleotide.

7. The genetic construct of claim 6, wherein N is A, T, G, or C.

8. The genetic construct of any one of claims 1 to 7, wherein the 3' splice site comprises a site located between two nucleotides containing G and N, wherein N is any nucleotide.

9. The genetic construct of claim 8, wherein N is C.

10. The genetic construct according to any one of claims 5 to 9, wherein the catalytic core comprises ACG, CU, or both.

11. The genetic construct according to any one of claims 1 to 10, wherein the excision scar comprises SEQ ID NO: 80 or SEQ ID NO:

82.

12. The genetic construct according to any one of claims 1 to 10, wherein the excision scar comprises SEQ ID NO: 81 or SEQ ID NO:

83.

13. The genetic construct according to any one of claims 1 to 11, wherein the excision scar comprises SEQ ID NO: 80 and SEQ ID NO:

82.

14. The genetic construct according to any one of claims 1 to 10 and 12, wherein the excision scar comprises SEQ ID NO: 81 and SEQ ID NO:

83.

15. The genetic construct according to any one of claims 1 to 14, wherein the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 3' intron sequences selected from Table 4 (SEQ ID NO: 11–22).

16. The genetic construct according to any one of claims 1 to 4 and 15, wherein the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 5' intron sequences selected from Table 4 (SEQ ID NO: 23-30).

17. The genetic construct according to any one of claims 1 to 4, wherein the 5' intron sequence and / or the 3' intron sequence are derived from the cyanobacterium *Anabaena spp.* tRNA LEU Introns.

18. The genetic construct of claim 17, wherein the 5' splice site comprises a site located between two nucleotides containing U and N, wherein N is any nucleotide.

19. The genetic construct of claim 18, wherein N is A.

20. The genetic construct of any one of claims 17 to 19, wherein the 3' splice site comprises a site located between two nucleotides containing G and N, wherein N is any nucleotide.

21. The genetic construct of claim 20, wherein N is C.

22. The genetic construct of any one of claims 17 to 21, wherein the catalytic core comprises GAG, CC, or both.

23. The genetic construct of any one of claims 17 to 22, wherein the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 3' intron sequences in Table 4, such as SEQ ID NO: 14 or 20.

24. The genetic construct of any one of claims 17 to 23, wherein the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 5' intron sequences in Table 4, such as SEQ ID NO: 25 or 29.

25. The genetic construct according to any one of claims 1 to 4, wherein the 5' intron sequence and the 3' intron sequence are derived from the genus *Vibrio zonatogenes*. tRNA LEU Introns.

26. The genetic construct of claim 25, wherein the 5' splice site comprises a site located between two nucleotides containing U and N, wherein N is any nucleotide.

27. The genetic construct of claim 26, wherein N is A.

28. The genetic construct of any one of claims 25 to 27, wherein the 3' splice site comprises a site located between two nucleotides containing G and N, wherein N is any nucleotide.

29. The genetic construct of claim 28, wherein N is G.

30. The genetic construct of any one of claims 25 to 29, wherein the catalytic core comprises GAG, CC, or both.

31. The genetic construct of any one of claims 25 to 30, wherein the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 3' intron sequences in Table 4, such as SEQ ID NO: 13 or 19.

32. The genetic construct of any one of claims 25 to 31, wherein the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 5' intron sequence in Table 4 (e.g., nucleotide sequence AAA).

33. The genetic construct according to any one of claims 1 to 4, wherein the 5' intron sequence and the 3' intron sequence are derived from introns of 26S ribosomal RNA from Tetrahymena thermophila.

34. The genetic construct of claim 33, wherein the 5' splice site comprises a site located between two nucleotides containing U and N, wherein N is any nucleotide.

35. The genetic construct of claim 34, wherein N is A.

36. The genetic construct of any one of claims 33 to 35, wherein the 3' splice site comprises a site located between two nucleotides containing G and N, wherein N is any nucleotide.

37. The genetic construct of claim 36, wherein N is U.

38. The genetic construct of any one of claims 33 to 37, wherein the catalytic core comprises a catalytic core selected from Table 5.

39. The genetic construct of any one of claims 33 to 38, wherein the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 3' intron sequence in Table 4, such as SEQ ID NO: 12 or 18.

40. The genetic construct of any one of claims 33 to 39, wherein the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the 5' intron sequence in Table 4, such as SEQ ID NO: 24 or 28.

41. A genetic construct comprising the following formula: 5'-HAEB-H'-3', Wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence (IGS), and H' contains a 3' homologous arm, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site, wherein the genetic construct is capable of forming a circular RNA, wherein the circular RNA does not contain any exon sequence ("excision scar") derived from the genome from which the 5' intron sequence and the 3' intron sequence are derived, and wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 113 or 123.

42. A genetic construct comprising the following formula: 5'-HAEB-H'-3', Wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence (IGS), and H' contains a 3' homologous arm, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site, wherein the genetic construct is capable of forming a circular RNA, wherein the circular RNA does not contain any exon sequence ("excision scar") derived from the genome from which the 5' intron sequence and the 3' intron sequence are derived, and wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 114 or 124.

43. A genetic construct comprising the following formula: 5'-HAEB-H'-3', Wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence (IGS), and H' contains a 3' homologous arm, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site, wherein the genetic construct is capable of forming a circular RNA, wherein the circular RNA does not contain any exon sequence ("excision scar") derived from the genome from which the 5' intron sequence and the 3' intron sequence are derived, and wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 115 or 125.

44. A genetic construct comprising the following formula: 5'-HAEB-H'-3', Wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence (IGS), and H' contains a 3' homologous arm, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site, wherein the RNA construct is capable of forming a circular RNA, wherein the circular RNA does not contain any exon sequence ("excision scar") derived from the genome from which the 5' intron sequence and the 3' intron sequence are derived, and wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 116 or 126.

45. A genetic construct comprising the following formula: 5'-HAEB-H'-3', Wherein H contains a 5' homologous arm, A contains a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence (IGS), and H' contains a 3' homologous arm, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site, wherein the genetic construct is capable of forming a circular RNA, wherein the circular RNA does not contain any exon sequence ("excision scar") derived from the genome from which the 5' intron sequence and the 3' intron sequence are derived, and wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 117 or 127.

46. ​​A genetic construct comprising the following formula 5'-HAEB-H'-3', Wherein, H contains a 3' homologous arm, A contains a 3' homologous arm and a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence, and H' contains a 5' homologous arm. The 3' intron sequence contains a catalytic core. The 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site. The 5' intron sequence and / or the 3' intron sequence are derived from the precursor tRNA of the cyanobacteria *Azotobacter spp.*, *Vibrio spp.*, or *Synechococcus*. LEU It may be derived from the 26S ribosomal RNA of Tetrahymena thermophila, or it may be able to form circular RNA.

47. The genetic construct of any one of claims 1 and 41 to 46, wherein the 5' intron sequence and / or the 3' intron sequence are derived from the tRNA of the cyanobacterium *Anabaena spp.* LEU .

48. The genetic construct of claim 47, wherein the 5' splice site comprises a site located between two nucleotides comprising U and any nucleotide (N), optionally A.

49. The genetic construct of claim 47 or 48, wherein the 3' splice site comprises a site located between two nucleotides comprising G and any nucleotide (N), optionally C.

50. The genetic construct of any one of claims 47 to 49, wherein the catalytic core comprises GAG, CC, or both.

51. The genetic construct of any one of claims 47 to 50, wherein the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 25 or 29.

52. The genetic construct of any one of claims 47 to 51, wherein the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 14 or 20.

53. The genetic construct of any one of claims 1 and 41 to 46, wherein the 5' intron sequence and / or the 3' intron sequence are derived from tRNAs of the cyanobacterium *Vibrio zoea*. LEU .

54. The genetic construct of claim 53, wherein the 5' splice site comprises a site located between two nucleotides comprising U and any nucleotide, optionally A.

55. The genetic construct of claim 53 or 54, wherein the 3' splice site comprises a site located between two nucleotides comprising G and any nucleotide, optionally C.

56. The genetic construct of any one of claims 53 to 55, wherein the catalytic core comprises GAG, CC, or both.

57. The genetic construct of any one of claims 53 to 56, wherein the 5' intron sequence comprises the nucleotide sequence of AAA.

58. The genetic construct of any one of claims 53 to 57, wherein the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 13 or 19.

59. The genetic construct of any one of claims 1 and 41 to 46, wherein the 5' intron sequence and the 3' intron sequence are derived from Synechococcus tRNA. LEU .

60. The genetic construct of claim 59, wherein the 5' splice site comprises a site located between two nucleotides comprising U and any nucleotide (N), optionally A.

61. The genetic construct of claim 59 or 60, wherein the 3' splice site comprises a site located between two nucleotides comprising G and any nucleotide (N), optionally C.

62. The genetic construct of any one of claims 59 to 61, wherein the catalytic core comprises GAG, CC, or both.

63. The genetic construct of any one of claims 59 to 62, wherein the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with a 5' intron derived from Synechococcus introns, as shown in SEQ ID NO: 140 or 143.

64. The genetic construct of any one of claims 59 to 63, wherein the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with a 5' intron derived from Synechococcus introns, as shown in SEQ ID NO: 140 or 143.

65. The genetic construct of any one of claims 1 and 41 to 46, wherein the 5' intron sequence and the 3' intron sequence are derived from the 26S ribosomal RNA of Tetrahymena thermophila.

66. The genetic construct of claim 65, wherein the 5' splice site comprises a site located between two nucleotides comprising U and any nucleotide (N), optionally A.

67. The genetic construct of claim 65 or 66, wherein the 3' splice site comprises a site located between two nucleotides comprising G and any nucleotide (N), optionally C.

68. The genetic construct of any one of claims 65 to 67, wherein the catalytic core comprises GAG, CC, or both.

69. The genetic construct of any one of claims 65 to 68, wherein the 5' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 24 or 28.

70. The genetic construct of any one of claims 65 to 69, wherein the 3' intron sequence comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 12 or 18.

71. The genetic construct of any one of claims 46 to 70, wherein the circular RNA does not contain excision scars.

72. The genetic construct according to any one of claims 1 to 71, wherein the circular RNA has reduced immunogenicity.

73. The genetic construct of claim 72, wherein the circular RNA has reduced immunogenicity compared to the circular RNA containing the excision scar.

74. A genetic construct comprising the following formula 5'-HAEB-H'-3', Wherein, H contains a 3' homologous arm, A contains a 3' homologous arm and a 3' intron sequence, E contains an exon sequence, B contains a 5' intron sequence containing an internal guide sequence, and H' contains a 5' homologous arm. The 3' intron sequence contains a catalytic core. The 3' end of A and the 5' end of E form a 3' splice site, and the 3' end of E and the 5' end of B form a 5' splice site. The 5' intron sequence and / or the 3' intron sequence are derived from the precursor tRNA of the cyanobacteria *Azotobacter spp.*, *Vibrio spp.*, or *Synechococcus*. LEU Or derived from 26S ribosomal RNA of Tetrahymena thermophila and capable of forming circular RNA, and said genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 118 or 128.

75. A genetic construct comprising the following formula: 5'-AE-3', Wherein A contains a 3' intron sequence, E contains an exon sequence, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splicing site, and wherein the genetic construct is capable of forming circular RNA.

76. A genetic construct comprising the following formula: 5'-AE-3', Wherein A contains a 3' intron sequence, and E contains an exon sequence, wherein the 3' intron sequence contains a catalytic core, wherein the 3' end of A and the 5' end of E form a 3' splice site, wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 119 or 129, and wherein the genetic construct is capable of forming a circular RNA.

77. A genetic construct comprising the following formula 5'-AE-3', Wherein A contains a 3' intron sequence containing a catalytic core (CC), and E contains an exon sequence containing a first portion (E'); a first homologous arm (HI'); a second portion (E''); a second homologous arm (HI''); and a third exon sequence containing a catalytic nucleotide (CN) (E'''); wherein the 3' end of A and the 5' end of E form a 3' splice site, wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 120 or 130, and wherein the genetic construct is capable of forming a circular RNA.

78. A genetic construct comprising the following formula 5'-AE-3', Wherein A contains a 3' intron sequence containing a catalytic core (CC), and E contains an exon sequence containing a first portion (E'); a first homologous arm (HI'); a second portion (E''); a second homologous arm (HI''); and a third exon sequence containing a catalytic nucleotide (CN) (E'''); wherein the 3' end of A and the 5' end of E form a 3' splice site, wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 121 or 131, and wherein the genetic construct is capable of forming a circular RNA.

79. A genetic construct comprising the following formula 5'-AE-3', Wherein H contains a 3' homologous arm, A contains a 3' intron sequence containing a catalytic core (CC), and E contains an exon sequence containing a first portion (E'); a first homologous arm (HI'); a second portion (E''); a second homologous arm (HI''); and a third exon sequence containing a catalytic nucleotide (CN) (E'''); wherein the 3' end of A and the 5' end of E form a 3' splice site, wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 122 or 132, and wherein the genetic construct is capable of forming a circular RNA.

80. A genetic construct comprising the following formula 5'-AE-3', Wherein A comprises a 3' intron sequence containing a catalytic core (CC), and E comprises an exon sequence containing a first portion (E'); a first homologous arm (HI'); a second portion (E''); a second homologous arm (HI''); and a third exon sequence containing a catalytic nucleotide (CN) (E'''); wherein the genetic construct comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 181, and wherein the genetic construct is capable of forming a circular RNA.

81. A genetic construct comprising the following formula 5'-BAE-3', Wherein A contains a 3' intron sequence, 3S contains a 3' splice site, E contains an exon sequence, B contains a 5' intron sequence, and 5S contains a 5' splice site, wherein the 3' intron sequence contains a catalytic core, wherein BA contains an intact intron, wherein the genetic construct contains a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with SEQ ID NO: 182 or 183, and wherein the genetic construct is capable of forming a circular RNA.

82. The genetic construct according to any one of claims 1 to 81, wherein it is RNA.

83. The genetic construct according to any one of claims 1 to 82, wherein it is DNA.

84. The genetic construct of claim 83, further comprising a promoter.

85. The genetic construct of claim 84, wherein the promoter comprises a T7 promoter, a T3 promoter, or a Sp6 promoter.

86. The genetic construct according to any one of claims 1 to 85, further comprising IRES.

87. The genetic construct of any one of claims 1 to 86, wherein the exon sequence further comprises a polyA-binding protein binding site at its 5' end.

88. The genetic construct according to any one of claims 1 to 87, wherein the 5' homologous arm comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to SEQ ID NO: 72 (CCGTCGATTGTCCACTGGT).

89. The genetic construct according to any one of claims 1 to 88, wherein the 3' homologous arm comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences to SEQ ID NO: 73 (ACCAGTGGACAATCGACGG).

90. The genetic construct of any one of claims 1 to 89, wherein the exon sequence is connected to one or more untranslated regions at the 3' and / or 5' of the exon sequence.

91. The genetic construct of claim 90, wherein the one or more untranslated regions comprise IRES.

92. The genetic construct of claim 91, wherein the IRES comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 87-110 and 145-168.

93. The genetic construct of claim 92, wherein the IRES is linked to polyA.

94. The genetic construct of any one of claims 90 to 93, wherein the one or more untranslated regions comprise polyA.

95. The genetic construct of claim 94, wherein the exon sequence is linked to polyA.

96. The genetic construct of any one of claims 93 to 95, wherein the polyA comprises a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 85 or 86.

97. The genetic construct of any one of claims 90 to 96, wherein the one or more untranslated regions further comprise spacer sequences.

98. The genetic construct of claim 97, wherein the spacer region comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with any of SEQ ID NO: 74-75 and 78-79.

99. The genetic construct of any one of claims 1 to 98, wherein the exon sequence encodes a protein.

100. The genetic construct of claim 99, wherein the circular RNA exhibits increased expression of the protein, prolonged expression of the protein in vitro and / or in vivo, decreased activation of immune signaling pathways, increased immune activation, or any combination thereof.

101. The genetic construct of claim 99 or 100, wherein the protein comprises a therapeutic protein.

102. The genetic construct of any one of claims 99 to 101, wherein the protein comprises a cytokine, a receptor, a ligand, an immunomodulator, a growth factor, an antigen, an RNA-binding domain, or any combination thereof.

103. The genetic construct of any one of claims 99 to 102, wherein the protein comprises an antibody, a fusion protein, or any combination thereof.

104. The genetic construct according to any one of claims 99 to 103, wherein the proteins comprise CD3, CD4, CDS, CD19, CD20, CD22, CD30, CD34, HER2, HER3, HER4, LFA-I, MoI, p150, 95, VLA-4, ICAM-I, VCAM, αv / β3 integrin, human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), NGF-β, platelet-derived growth factor (PDGF), fibroblast growth factor, epidermal growth factor (EGF), transforming growth factor (TGF), insulin-like growth factor-I and-II (IGF-I and IGF-II), des(l-3)-IGF-I (Brain IGF-1), Factor VIII, Tissue Factor, von Willebrand Factor, Protein C, α-1-antitrypsin, plasminogen activator such as urokinase and tissue plasminogen activator ("t-PA"), bufotoxin, thrombin, thrombopoietin, M-CSF, GM-CSF, G-CSF, flk2 / flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptor, thrombopoietin receptor ("TPO-R", "c-mpl"), glucagon receptor, interleukin receptor, interferon receptor, T cell receptor, stem cell factor receptor, OX40L, bone-derived neurotrophic factor (BDNF), neurotrophic factor-3,-4,-5 or-6 (NT-3, NT-4, NT-5, or NT-6), relaxin A chain, relaxin B chain, pro-relaxin, interleukins and interleukin receptors such as IL-8 receptor including IL-1 to IL-33 and IL-1 to IL-33 receptors, viral antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-α and-β, enkephalin, RANTES (regulatory activation of normal T-cell expression of secretory factors), mouse gonadotropin-related peptide, DNase, inhibin, activin, PUF domain, Cas protein, or any combination thereof.

105. A composition comprising a genetic construct and GTP as described in any one of claims 1 to 104.

106. The composition of claim 105, wherein the amount of GTP is sufficient to cleave the 5' intron sequence and the 3' intron sequence from the genetic construct.

107. A method for preparing circular RNA, comprising contacting GTP with a genetic construct as described in any one of claims 1 to 104.

108. A circular RNA prepared by the method of claim 107.

109. A method of treating a disease or condition in a subject in need, comprising administering the circular RNA as described in claim 108 to the subject.

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